EP0996708A1 - Modele de nematode transgenique de maladies neurologiques a repetition par triplet - Google Patents

Modele de nematode transgenique de maladies neurologiques a repetition par triplet

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Publication number
EP0996708A1
EP0996708A1 EP98935611A EP98935611A EP0996708A1 EP 0996708 A1 EP0996708 A1 EP 0996708A1 EP 98935611 A EP98935611 A EP 98935611A EP 98935611 A EP98935611 A EP 98935611A EP 0996708 A1 EP0996708 A1 EP 0996708A1
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Prior art keywords
nematode
nucleic acid
trend
protein
transgenic
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German (de)
English (en)
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EP0996708A4 (fr
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Anne C. Hart
Peter Faber
Marcy Macdonald
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General Hospital Corp
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General Hospital Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/641—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0335—Genetically modified worms
    • A01K67/0336—Genetically modified Nematodes, e.g. Caenorhabditis elegans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to transgenic animal models of triplet repeat neurological diseases such as Huntington's disease.
  • Transgenic animal models have recently become valued tools in the elucidation of human disease processes as well as in the characterization of therapeutic drugs for disease treatment.
  • Huntington's disease a dominant inherited, untreatable neurodegenerative disorder, a particular need for accurate animal models exists.
  • Huntington's disease The most characteristic feature of Huntington's disease is its peculiar movement disorder which begins subtly and progresses to exaggerated dance-like motions that eventually consume the entire body. Huntington's disease occurs equally in both sexes and is found in all races, but is diagnosed most frequently (i.e., about 1 in 10,000) in people of Western European descent. Although symptoms may begin at any age, they generally appear initially between the ages of 30 and 55, and they progressively worsen until death 12-18 years later. The clinical progression of Huntington's disease is paralleled by neuronal degeneration in the brain.
  • Huntington's disease is the loss of medium spiny GABAergic projection neurons in a gradient progressing along posteroanterior, dorso ventral, and mediolateral axes of the caudate nucleus.
  • signs of neuronal dysfunction are evident in recurved dendritic endings and changes in spine density, shape, and size.
  • the disorder eventually destroys the architecture of the caudate nucleus and the adjacent putamen, although extensive cell loss also occurs in other regions of the basal ganglia and in the deep layers of the cerebral cortex. Overall brain weight may be reduced by 25% or more.
  • the proximate cause of the neuronal dysfunction and death is not yet known, it is ultimately due to the presence of a mutant gene located near the telomere of the chromosome 4 short arm.
  • the Huntington's disease mutation discovered in 1993, occurs in the first exon of a 67-exon gene encoding a large novel protein. All Huntington's disease patients have an expansion in a sequence of consecutive CAG codons that lengthens the stretch from the 10 to 34 repeat units seen on normal chromosomes to more than 36 repeat units. Since discovery of the Huntington's disease gene, it has remained unclear how the expanded CAG repeat causes specific neuronal loss.
  • the invention features a transgenic nematode that includes a TREND
  • triplet repeat neurological disease -associated nucleic acid sequence operably linked to a promotor allowing expression in neurons.
  • the expression of the TREND-associated nucleic acid expression is limited to all or a part of the nematode's nervous system;
  • the TREND- associated nucleic acid expresses a TREND protein (for example, the human Huntingtin protein); the animal exhibits progressive neuronal dysfunction (for example, loss of the ASH neurons or M4 neuron functions in the nematode); dysfunction is seen in at least one nerve cell; at least 40% of the animals in a given population show nerve cell dysfunction;
  • the nematode further includes a homozygous loss of function mutation in a gene encoding a cysteine protease, more preferably, in a gene encoding a caspase, and, most preferably, in the ced-3 gene; and the nematode is C. elegans, C. vulgaris, or C. b ⁇ ggsae.
  • the invention features a transgenic nematode exhibiting time-dependent loss of neurons.
  • the nematode exhibits no substantial non-neuronal cell dysfunction prior to the onset of neuronal cell dysfunction; the degree of neuronal cell dysfunction progressively worsens with the age of the nematode; the nematode exhibits a decreased number of neurons (preferably, loss of at least one neuron, more preferably, two neurons relative to the number of neurons present at the LI stage); the animal is capable of transferring the neuronal cell dysfunction trait to its offspring; the nematode expresses a TREND-associated nucleic acid (for example, a nucleic acid which encodes the Huntingtin protein, and preferably a human Huntingtin protein (for example, at least the N- terminal 17 amino acids of the human Huntingtin protein covalently bonded to at least 119 additional glutamines, more preferably at least 150 glutamines, and most preferably 173 additional glutamines)); the TREND nucleic acid is a fragment of a naturally-occurring TREND gene including
  • the invention features a method of producing a transgenic nematode that exhibits progressive neuronal cell death, involving
  • Huntingtin nucleic acid for example, the nucleic acid encoding at least the N-terminal 17 amino acids of the human Huntingtin protein followed by at least 119, more preferably at least 150, and most preferably at least 173, CAG repeats encoding glutamine (i.e., 96 or 150 additional CAG repeats relative to the naturally-occurring 23 CAG repeat stretch which occurs in the non-disease causing wild-type Huntingtin gene beginning at the nucleic acid encoding amino acid 18)); the expressible genetic construct includes a neuron-specific regulatory region (e.g., the osm-10 regulatory region or a regulatory region which directs expression in the M4 pharynx neuron); the nucleic acids are present in an array having a marker gene (e.g., lin-15) suitable for microinjection into nematodes; the nematode further includes a homozygous loss of function mutation in a gene encoding a cysteine protease, more
  • the invention features a method for expressing a nucleic acid sequence of interest in a nervous system-specific manner, involving operably linking the nucleic acid sequence of interest to an osm-10 regulatory region, the nucleic acid sequence of interest being positioned in a region that, in the naturally-occurring osm-10 gene, includes all or a part of the OSM-70 protein-coding region and the nucleic acid sequence of interest being expressed under the control of the osm-10 regulatory region.
  • the expression occurs in C. elegans;
  • the nucleic acid sequence of interest is a TREND-associated nucleic acid (for example, having the coding sequence for a Huntingtin protein, for example, the N-terminal 17 amino acids of human Huntingtin protein followed by at least 119 CAG repeats encoding glutamines, more preferably at least 150 repeats, or most preferably at least 173 repeats);
  • the expression occurs in non-essential neurons; and all or a part of the protein coding region of osm-10 has been deleted, for example, the C-terminal region has been deleted, more preferably amino acids 18 to 419.
  • the invention features a transgene including (a) a nucleic acid sequence of interest linked to (b) an osm-10 regulatory region, the nucleic acid sequence of interest being positioned in a region that, in the naturally-occurring osm-10 gene, includes all or a part of the OSM-70 protein coding region and the nucleic acid sequence of interest being expressed under the control of the osm-10 regulatory region.
  • the nucleic acid sequence of interest is a TREND- associated nucleic acid (for example, encoding a Huntingtin protein, for example, encoding at least the N-terminal 17 amino acids of human Huntingtin protein followed by at least 119 CAG repeats, more preferably at least 150 repeats, and most preferably at least 173 repeats); and expression occurs in non-essential neurons.
  • TREND- associated nucleic acid for example, encoding a Huntingtin protein, for example, encoding at least the N-terminal 17 amino acids of human Huntingtin protein followed by at least 119 CAG repeats, more preferably at least 150 repeats, and most preferably at least 173 repeats
  • the invention features a method of testing a substance for efficacy in the treatment or prevention of a TREND-associated disease, involving exposing a nematode of the invention to the substance and determining the extent of the progressive nerve cell dysfunction exhibited by the nematode following substance exposure, a decrease in the progressive nerve cell dysfunction, relative to an unexposed nematode of the invention, indicating a substance useful for the treatment of a TREND-associated disease. Also included in the invention are substances identified by this method.
  • the nematode expresses a nucleic acid having a sequence encoding a Huntingtin protein (for example, a human Huntingtin disease protein, for example, at least the N-terminal 17 amino acids of the human Huntington protein, followed by at least 119, more preferably at least 150, and most preferably at least 173, glutamine residues; the nematode is C. elegans, C. vulgaris, or C.
  • a Huntingtin protein for example, a human Huntingtin disease protein, for example, at least the N-terminal 17 amino acids of the human Huntington protein, followed by at least 119, more preferably at least 150, and most preferably at least 173, glutamine residues
  • the nematode is C. elegans, C. vulgaris, or C.
  • the determining is done by assaying for loss or delay of loss of the ASH neurons; the assaying is done by dye filling (e.g., with the vital dyes such as Dil, DiO, or FITC); the assaying is done by use of a Green Fluorescent Protein (GFP) protein (preferably the GFP is covalently linked to the TREND polypeptide); the assaying is done by assaying for loss of ASH or M4 neuron dysfunction (e.g., by monitoring nose touch response or starvation); and the assaying is done at approximately seven days after hatching of the nematode or at the time when nerve cell dysfunction normally becomes measurable in the nematode.
  • GFP Green Fluorescent Protein
  • transgenic nematode any nematode which includes a nucleic acid sequence which is inserted by artifice into a cell and becomes a part of the genetic material of at least some of the cells of the nematode that develops from that cell, e.g., an ASH, M4, or CAN neuron or a CAN cell.
  • a transgene may be partly or entirely heterologous to the transgenic animal.
  • the transgene may be part of an extrachromosomal array, or may be integrated into the genome of the animal.
  • transgenic C. elegans nematodes represent a preferred embodiment of the invention, other transgenic nematodes include, without limitation, transgenic C. briggsae and C. vulgaris. Such transgenic nematodes may be constructed by standard techniques using the description herein and are included in the invention.
  • progressive nerve cell dysfunction is meant a loss of normal function of at least one neuron after hatching of the nematode, relative to a nematode lacking the transgene but otherwise genetically identical.
  • loss of function is measured by loss of dye filling, as described herein, or loss of GFP otherwise detectable when expressed from a genetic construction in a nematode lacking the TREND nucleic acid sequences, or by failure of the nematode to survive on a lawn of OP50 bacteria.
  • OSM-10 protein is meant a naturally-occurring polypeptide sequence encoded by a nucleic acid sequence capable of hybridizing to the nucleic acid sequence of Fig. 1 (SEQ ID NO: 1
  • the protein is the protein encoded by the osm-10 sequence of Genbank accession #U00037.
  • osm-10 protein coding region is meant a nucleic acid sequence capable of encoding an OSM-10 protein.
  • sequence is the coding sequence of the osm-10 gene provided in Fig. 1 (SEQ ID NO:l).
  • osm-10 regulatory region is meant a nucleic acid sequence capable of hybridizing to nucleic acids 12845-13737 of Fig. 1 (SEQ ID NO:l) under high stringency conditions and which is capable of directing nervous system-specific gene expression.
  • the sequence is the sequence of the osm-10 promoter shown in Fig. 1 as nucleic acids 12845-13734 or a fragment thereof.
  • TREND-associated disease is meant a neurological disease which correlates with an expanded trinucleotide repeat in a gene of symptomatic individuals.
  • the repeat may be a CAG repeat.
  • Huntington's disease spinal bulbar muscular atrophy (SBMA: Fischbeck, K.H. (1995), Proc. Assoc. Am. Physicians 107:228-230; Brooks, B.P. et al. (1995), Trends Neurosci. 1 ⁇ :459-461), dentatorubral-pallidoluysian atrophy (DRPLA: Burke, J.R. et al. (1994), Nat. Genet. 7:521-524; Nagafuchi, S. et al.
  • Psychiatry 59:449-450; Zhuchenko, O. et al. (1997), Nat. Genet. 11:62-68; Trottier, Y. et al. (1995), Nature 378:403-406; and Lindblad, K. et al. (1996), Genome Res. 6:965-971) are TREND diseases.
  • Non CAG triplet repeat diseases are also included.
  • TREND-associated disease phenotype is meant an increase in nerve cell dysfunction relative to a wild-type nematode.
  • the neurons are ASH neurons or the M4 neuron.
  • TREND-associated nucleic acid any nucleic acid sequence, regardless of length or post-translational modification, that has been, or will be, found to cause in whole or in part a TREND-associated disease or symptom. Included are human TREND genes, functional homologous from other species, and wild-type homologues modified with a sufficient number of trinucleotide repeats, as provided herein. Such nucleic acids include, without limitation, any naturally occurring TREND nucleic acid sequence having trinucleotide repeats of nucleic acid residues relative to the wild-type (non-disease causing) homology.
  • At least one triplet repeat associated with the appearance of the TREND disease, relative to the non-disease causing homology is added, more preferably at least 150, even more preferably at least 200, and most preferably more than 300.
  • the term includes nucleic acids which are fragments or precursors of the naturally occurring disease causing gene.
  • at least 500 nucleic acids flanking the trinucleotide repeat are present in the TREND-associated nucleic acid.
  • the nucleic acid may be from a CAG or non- CAG triplet repeat TREND gene.
  • a "TREND-associated protein" is an amino acid sequence encoded by a TREND- associated nucleic acid.
  • huntingtin protein is meant a protein encoded by nucleic acid capable of hybridizing at high stringency to nucleic acids 316-366 of Fig. 2 (SEQ ID NO:2) and which further includes at least 119, and more preferably at least 173, glutamine residues.
  • the first 17 amino acids of the polypeptide are the first 17 amino acids of the protein sequence provided in Fig. 3 (SEQ ID NO:3), and the glutamine residues are covalently linked to the C- terminus of the first 17 amino acids.
  • a "Huntingtin” gene is a nucleic acid sequence which is capable of hybridizing at high stringency conditions to nucleic acids 316-366 of Fig. 2 and which confers a nerve cell dysfunction phenotype when expressed in nerve cells (particularly the ASH neurons).
  • homology is meant a gene or protein having at least 60%, and more preferably 80%, identity to the human disease-causmg TREND nucleic acid over a stretch of at least 200 nucleic acids and which confers TREND-associated nerve cell dysfunction when expressed in a nerve cell (particularly in an ASH neuron when expressed under the control of the osm- 10 regulatory region).
  • high stringency conditions hybridization in 2X SSC at 40 °C with a
  • DNA probe length of at least 40 nucleotides.
  • high stringency conditions see Ausubel, F. et al., 1994, Current Protocols in Molecular Biology. John Wiley
  • neural cell-specific or “neuron-specific” is meant that expression of a nucleic acid sequence occurs substantially in a nervous system tissue (for example, the ASH, ASI,
  • the expression of the nucleic acid sequence in the nervous system tissue represents at least a 5 -fold, more preferably, a 10-fold, and, most preferably, a 100-fold increase over expression in non-nervous system tissue.
  • regulatory region is meant a sequence which is minimally necessary for directing transcription and, if appropriate, translation of an associated nucleic acid coding sequence.
  • the term may also include auxiliary sequences that mediate gene expression in response to an external or internal stimulus, for example, expression that is inducible (for example, by temperature or a chemical stimulus) or expression that is tissue-specific (for example, nervous system-specific) or developmental stage-specific.
  • "Regulatory region” sequences are generally located 5' (or “upstream") of the nucleic acid sequence encoding polypeptide, but may be located within or 3' (or “downstream") of the coding sequence.
  • introducing into a hermaphrodite is meant to encompass any method by which a transgene may be introduced into a nematode 's offspring including, without limitation, microinjection of the parent hermaphrodite with nucleic acids.
  • treatment of a TREND-associated disease is meant the ability to reduce, prevent, or retard the onset of any symptom associated with a TREND disease, particularly those resulting in progressive neuronal cell death.
  • operably linked or "in an expressive genetic construct” is meant that a nucleic acid sequence and a regulatory sequence(s) are connected in such a way as to permit expression of that nucleic acid sequence, e.g., osm-10, egl-l, or arrestin-l.
  • covalently bonded is meant joined either directly through a covalent bond or indirectly through another covalently bonded sequence.
  • the transgenic animals developing progressive nerve cell dysfunction described for the first time herein have the advantage of mimicking the progressive nerve cell dysfunction observed in patients with TREND diseases.
  • the progress of this critical symptom i.e., the loss of specific neuronal cells, is easily observable in the same animal or a population of animals hatched together over time.
  • this animal model is also useful for the testing of palliative therapies which delay the appearance of nerve cell dysfunction.
  • this invention provides a transgenic nematode model of TREND diseases with measurable nerve cell dysfunction, compounds may be screened to identify those which alleviate this loss, even absent knowledge of the symptom's underlying biological cause.
  • the associated neuropathological symptoms exhibited by the transgenic animal models described herein provide the unique advantage of allowing the investigation of the etiology of TREND diseases.
  • loss of ASH dye filling in the nematode may be correlated with molecular events associated with the etiology of the disease.
  • treatments which are shown to diminish the occurrence of neuronal cell death may be tested for their ability to selectively improve certain pathological symptoms in higher organisms or patients.
  • nematode model Significant additional advantages of the nematode model are the short generation time, the ability to genetically manipulate large numbers of animals, and the ability to harvest and accurately stage large numbers of animals for high throughput screening.
  • Another advantage of this invention is the ease with which these transgenic animals are bred to produce identical transgenic offspring. Also, because these transgenic animals may be bred as readily as control animals and because they produce similar members of offspring, the animals of the invention may be generated in sufficient quantity to make them widely and rapidly available to researchers in this field. With respect to the construct used to generate the particular transgenic animals described herein, the use of the osm-10 promoter also provides additional advantages. First, this construct is active only in neuronal tissues of the nematode (most particularly the ASH, ASI, PHA, and PHB neurons). In addition, the osm-10 gene has the advantage of being expressed in non-essential neurons of the nematode.
  • the deletion of a portion of the OSM- 10 protein coding region also has the advantage of providing an ideal site for transgene insertion, for example, for the insertion of the nucleic acids encoding the N-terminal 17 amino acids of the Huntingtin gene plus sufficient glutamine residues to produce progressive nerve cell dysfunction.
  • preferred transgenic animals according to the invention include nucleic acid encoding the first 17 amino acids of the human Huntingtin gene plus at least 119 glutamines residues, more preferably at least 173 glutamine residues carboxy-terminal to the 17 N-terminal Huntingtin amino acids.
  • Fig. 1 is a schematic drawing showing a portion of the osm-10 gene, including the regulatory region.
  • Fig. 2 is a schematic diagram of the Huntingtin gene.
  • Fig. 3 is a schematic diagram of the huntingtin protein sequence
  • Huntington's disease is an autosomal dominant, neurodegenerative disorder caused by expansion of a poly-glutamine domain in the N-terminus of the huntingtin protein.
  • the mechanism by which the mutant Huntingtin gene causes neurodegeneration is unknown.
  • the length of the poly-glutamine stretch in the huntingtin protein is inversely proportional to the age of onset in humans.
  • the C. elegans model permits large scale screening of drugs and chemical therapies for prevention and treatment of Huntington' s disease and other triplet repeat neurodegenerative disorders (including spinocerebellar ataxia I and II, dentatorubropallidoluysian atrophy, and spinal bulbar muscular atrophy, as well as non-CAG triplet repeat diseases), and also for other poly-glutamine-mediated diseases in non-neuronal tissues.
  • the C. elegans model also permits the identification and analysis of the mechanism of neuronal death mediated by poly-glutamine proteins and the identification of genes and proteins involved in this neuropathology.
  • transgenic animals of the invention are described in detail below. In general, these animals are produced by first creating a construct that includes a promoter that directs nervous system-specific expression linked to a gene associated with a TREND disease.
  • This construct is amplified in bacterial cells, purified, and injected into hermaphrodite worms. Resulting offspring that have incorporated the foreign gene into their cells are identified using a linked marker present on the array. From these founder animals, several distinct animal lines are produced. By crossing with wild-type nematodes or nematodes having other mutations of interest, additional strains may be constructed which facilitate screening.
  • transgenic animals described herein exhibit progressive neuronal cell death in the absence of changes in the environment or other parameters relative to controls lacking the
  • TREND construct or having the first 17 amino acids of the huntingtin gene homology from an (undiseased) individual (i.e., the wild-type gene).
  • mutant huntingtin proteins expressed in the transgenic nematodes included the first 17 amino acids of the huntingtin protein linked to a 150 (Q150), 95 (Q95), 23 (Q23), or
  • Dye filling requires an intact sensory cilium exposed to the environment. Thus, to determine whether the defective dye filling resulted from ASH death or a defect in ASH sensory cilium, neuronal viability was assessed using anti-endogenous OSM antiserum to test for OSM expression. In both Q150-expressing and control animals, essentially all ASH neurons (98%) stained for OSM, and, therefore, were viable. Accordingly, the Q150- mediated dye filling defect was caused by degeneration or truncation of the ASH sensory cilium, rather than cell death. No dye filling defects were observed for the Q95, Q23, or Q2 nematodes at any age.
  • Co-expression of huntingtin fragments with OSM-10::GFP allowed rapid discrimination between degeneration and cell death, avoiding the problem of underscoring the huntingtin fragment-mediated defects because of mosaicism.
  • Expression of OSM- 10::GFP sensitized neurons to the effects of the huntingtin fragments, resulting in cell death as well as an increased rate of dye defects as compared to non-sensitized neurons.
  • those that expressed OSM-10::GFP were scored as dye filling defective, whereas those that failed to express OSM-10::GFP were scored as dead.
  • Q150 co-expression resulted in a dye filling defect in 3% of neurons at 3d, which increased to 26%) at 8d.
  • Q150 also caused cell death in 2%> of 3d ASH neurons, increasing to 10% at 8d. This death rate was supported by OSM- 10 antiserum results using rigorous scoring parameters, which demonstrated that at least 6% of the Q150 expressing ASH neurons were dead.
  • Co-expression of the huntingtin fragments with OSM-10::GFP allowed unequivocal identification of the ASH in fixed nematodes.
  • the huntingtin fragments were visualized using the huntingin antiserum HF1 at 3d and 8d. Expression of huntingtin fragments also resulted in cytoplasmic aggregation.
  • the huntingtin fragments were visualized throughout the sensitized ASH cytoplasm, including processes and axons.
  • the Q2, Q95, and Q150-expressing neurons showed diffuse staining of fragments. Furthermore, the Q150 fragments were stained in 20% of the 3d ASH neurons.
  • Q95-expressing neurons demonstrated low level aggregation, and 90% of Q150-expressing neurons demonstrated large protein aggregates.
  • the huntingtin fragments were visible using Nomarski illumination and visible light.
  • the effects of huntingtin fragment expression were also assessed using the ASH- mediated response to nose touch as a behavioral assay.
  • the nose touch response was reduced to 64% and 41% of control, respectively.
  • animals lacking ASH neurons exhibited a nose touch response reduced to 35% of control. No behavioral effect was observed in nematodes expressing either Q23 or Q2 in combination with OSM-10::GFP,
  • ced-3 encodes a caspase, a cysteine protease from the ICE family.
  • a caspase a cysteine protease from the ICE family.
  • rtEx[7B,C,D] Q150 expression resulted in a 7% dye filling defect, a 4-fold decrease when compared to the 27% defect exhibited in Ql 50-expressing nematodes with the wild type ced-3.
  • OSM-10::GFP expression resulted in dye filling defects in 1% of neurons at 3d and 26% at
  • the OSM-10::GFP dye filling defects were relatively independent of ced-3 gene product function. Loss of function ced-3 mutations eliminated only 24% of the OSM-10::GFP- mediated defect (54% in wild-type animals compared to 41% in ced-3 mutant nematodes).
  • the dye filling defect virtually disappeared at lower levels of expression with 1% of neurons exhibiting the defect in 8d wild-type nematodes.
  • ced-3 function was assessed.
  • Expression of an extrachromosomal array, rtEx53A caused cell death in 10% of wild-type ced-3 nematodes, yet caused no cell death in the ced-3 mutants.
  • the extrachromosomal array was crossed back in a wild-type background. This restored the rate of cell death to that observed originally in the wild-type nematodes.
  • Q150 protein aggregation also required ced-3 function, at least in part.
  • Ced-3 wild-type nematodes expressing the rtEX53A array formed aggregates in 86% of neurons. In contrast, only 50% of neurons formed protein aggregates in ced-3 mutant nematodes. Following return to a wild-type background, protein aggregation was restored to
  • CED-3 was the primary effector of apoptosis in C. elegans.
  • ced-3 mutant animals the ability of Q150 to cause an ASH dye filling defect was significantly reduced.
  • cell death was completely dependent on ced-3 function. The role of apoptosis in cell death has not been reported for mouse models.
  • NI neurodegenerative diseases
  • C. elegans C. elegans
  • cellular dysfunction and cell death occurred in the absence of NI, suggesting that NI played no active role in the disease process.
  • NI were observed in SCA7 patient brains in regions unaffected by disease and in all expressing tissues in the SCA3/Drosophila model, even those unaffected by SCA3 expanded fragment expression.
  • NI we observed large and small cytoplasmic aggregates of Q 150 and Q95.
  • the wild-type lengths, Q23 and Q2 did not aggregate, indicating that aggregation did not result from ectopic expression of the huntingtin fragment.
  • Non-nuclear polyQ deposits have been observed in dystrophic neurites for HD. Aggregate formation was independent of ced-3 function, at least in part, given that aggregation was only partially blocked in ced-3 mutants. This indicated that blocking the disease-causing effects of polyQ fragments still led to significant protein deposits.
  • the osm-10 promoter/Huntington's disease construct used to generate animals according to the invention was produced as follows.
  • osm-10 gene was cloned as described below. Constructs were then generated which included nucleotides 12845 to the BamHl site at nucleotides 13894-13899 of Fig. 1
  • SEQ ID NO: 1 (from CT20H4). This fragment includes the osm-10 promoter region and a sequence that encodes 17 amino acids from the N-terminus of the OSM- 10 protein. Huntingtin-encoding nucleic acids were inserted 3' of the OSM- 10 nucleic acid sequences. These constructs were as follows: (i) the control construct (HD+) having nucleic acids (Fig. 2, SEQ ID NO:2) encoding amino acids 1-17 of the non-disease causing (wild-type) human
  • Huntingtin protein (Fig. 3, SEQ ID NO: 3); and (ii) the test constructs having an additional 23 (HD23), 95 (HD95), or 150 (HD150) CAG repeats inserted into the polyglutamine- encoding stretch (as amino acids 18-40) of the huntingtin sequence present in the HD+ construct.
  • These nucleic acid sequences were cloned into the vector described in Fire et al. (Gene (1990) 95_: 189-198) and used to microinject hermaphrodite nematodes. Transgenic offspring were then isolated and assayed with vital dyes for neuron survival or dysfunction. This general technique is readily adaptable for use with other TREND genes or other vector systems.
  • Fluorescein isothiocyanate (FITC). DiO. and Dil starting protocol A stock dye solution containing 20 mg/ml 5-fluorescein isothiocyanate Dil or DiO in dimethylformamide was stored at -7°C. If kept dry, this stock solution is stable.
  • worms may instead be soaked in a liquid solution of dye and then placed on plates lacking dye, as with the plate method.
  • This liquid protocol reduced interfering dye fluoresence from the intestine by staining and destaining the animals in ice-cold M9 buffer. Specifically, the chilled animals do not feed and hence no dye enters the intestine.
  • animals were washed free of bacteria in M9 buffer alone, stained for 4 hours in cold M9 buffer containing 0.4 mg/ml FITC, washed three times in cold M9 buffer, and then transferred to a seeded agar plate at room temperature.
  • Sensory neurons can be divided into two groups, neurons like photoreceptor cells that normally detect one type of stimulus and neurons, termed polymodal neurons, that respond to more than one type or mode of stimulation.
  • Polymodal neurons that respond to more than one type or mode of stimulation.
  • C. elegans can distinguish between nose touch and other stimuli detected by ASH; habituation to nose touch eliminates 60% of the response to nose touch, but leaves intact response to high osmolarity and volatile repellents.
  • the ASH neurons express both putative mechanoreceptors and chemoreceptors (e.g.,
  • the osm-10 gene was found to map between mec- 14 and lin-39 and was identified based on phenotypic rescue.
  • the nucleic acid sequence is shown in Fig. 1.
  • the protein is
  • nl602 mutation changes an E to a K at amino acid 199, and fulfills genetic criterion for a null allele.
  • osm- ⁇ 0::GFF reporter constructs and polyclonal sera osm-1 appeared to be uniformly distributed in the cytoplasm of ASH, ASI, PHA, and PHB sensory neurons. Laser ablation experiments suggest that ASI, PHA, and PHB are not required for osmotic avoidance.
  • nl602 causes the substitution of a lysine for a glutamic acid in the putative extracellular domain. Although nl602 acts as a null allele in genetic tests, it may correspond to a partial loss of function. Insertions of the GFP coding sequence in the rescue construct eliminated osm-10 rescue activity, but the resulting reporter constructs consistently had GFP expression in four classes of sensory neurons: ASH, ASI, PHA, and PHB. All four classes of neurons have exposed, ciliated, sensory endings (ASH and ASI in the anterior; PHA and PHB in the posterior).
  • the screening methods of the invention involve screening any number of compounds for therapeutically active agents by employing any number of in vivo experimental systems. Exemplary methods useful for the identification of such compounds are detailed below. The methods of the invention simplify the evaluation, identification, and development of active agents for the treatment or prevention of TREND-associated conditions, such as nerve dysfunction and nerve cell loss.
  • the screening methods provide a facile means for selecting natural product extracts or compounds of interest from a large population which are further evaluated and condensed to a few active and selective materials. Constituents of this pool are then purified and evaluated in the methods of the invention to determine their activities.
  • novel drugs for the treatment of TREND-associated conditions are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art.
  • test extracts or compounds are not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.
  • Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA).
  • natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods.
  • any library or compound is readily modified using standard chemical, physical, or biochemical methods.
  • the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having TREND disease preventative or palliative activities.
  • the same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using a mammalian TREND disease model.
  • transgenic C. elegans having a TREND nucleic acid e.g., C. elegans containing mutations described herein, such as C. elegans having the HD150 construct described above
  • C. elegans having the HD150 construct described above are cultured in wells of a microtiter plate, facilitating the semiautomation of manipulations and full automation of data collection.
  • compounds that prevent or delay the loss of dye filling of the ASH neurons or prevent starvation due to dysfunction of the M4 neuron are considered useful in the invention.
  • Such compounds are identified by their effect on preventing or delaying the nerve cell dysfunction in C. elegans strains carrying those constructs of the invention (as described above).
  • nematodes bearing the TREND HD150 construct lose the ability to take up dye in the ASH neurons at 7-8 days after hatching.
  • visual screening or automated screening for the presence or absence of a suitable dye compound at various times after hatching may be used to identify compounds which delay or prevent the loss of dye filling.
  • test compound or extract can be inoculated to assess the dosage effect.
  • Control wells are incubated in the absence of a test compound or extract. Plates are then incubated at 25 °C. After the appropriate periods of time, e.g., 2 to 6 days, nematodes from wells are examined for the presence of neuronal dye filling. A delay in loss of dye filling is taken as an indication that the test compound or extract may be effective at inhibiting a TREND-associated condition, and therefore is considered useful in the invention.
  • a nerve cell dysfunction may arise from expression of a TREND nucleic acid in the M4 neuron.
  • the worms starve, unless given special bacteria to feed upon (A very et al, Neuron (1984) 3:473-485). Accordingly, nematodes which are exposed to a compound and which survive in the presence of the compound beyond the period of survival for unexposed worms also identify a useful compound.
  • C. elegans worms expressing TREND nucleic acids may be utilized in assays to obtain additional information on the mode of action of the test compound in the pathogenic TREND disease.
  • neural function can be assessed using the nose touch assay, as previously described in Kaplan and Horvitz (Proc. Natl. Acad. Sci. (1993) 90: 2227-2231). Briefly, nematodes are placed on an agar surface and an object of appropriate thickness, e.g., a hair, is placed on the surface such that the nematode moves to contact the foreign object with its nose. While wild-type nematodes respond to this noxious stimuli by immediately backing up, mutant nematodes expressing huntingtin fragments fail to respond or have a reduced response to this stimuli. If desired, the effect of additional mutations, e.g., ced-3, on the huntingtin fragment can be assessed.
  • additional mutations e.g., ced-3
  • the assay can be conducted after various concentrations of test compounds or extracts are administered to the nematode. If the compound or extract modulates the behavioral response in huntingtin mutant nematodes, e.g., increases the nose touch response towards the wild-type level, then the compound or extract may be effective in inhibiting a TREND-associated condition.
  • reporter gene constructs such as a green fluorescent protein (GFP) sequence under the control of the OSM- 10 promoter or another promoter allowing expression in a nerve cell affected by expression of a TREND nucleic acid.
  • GFP green fluorescent protein
  • C. elegans strains also expressing a TREND nucleic acid (e.g., the HD150 construct).
  • TREND nucleic acid e.g., the HD150 construct
  • TREND nucleic acid whether isolated from a diseased individual or modified from the wild-type gene, is accomplished according to standard methods, and, if desired, such nucleic acid may be operatively linked to a gene promoter obtained from C. elegans, such as the osm-10 regulatory region.
  • the invention involves the use of a reporter gene that is expressed under the control of a C. elegans nerve-specific promoter.
  • Other equivalent promoter elements may also be used in the invention.
  • the promoter element is cloned upstream of any standard reporter gene, e.g., the luciferase or green fluorescent protein (GFP) reporter genes.
  • the reporter gene construct is introduced into an appropriate nematode host (e.g., C. elegans) and an analysis of reporter gene activity is determined in the presence and absence of a candidate compound according to any standard method known in the art.
  • Such reporter gene (and host cell systems) are useful for screening for drugs that prevent or delay TREND-associated nerve cell loss or dysfunction.
  • useful therapeutic compounds include those which prevent or delay loss of ASH dye filling or the loss of M4 function as measured by starvation.
  • ASH dye filling or viability are measured following the addition of candidate antagonist molecules to a culture medium of nematodes containing the TREND nucleic acids (preferably a staged culture).
  • candidate antagonists may be directly administered to nematodes and used to screen for their effects on nerve cells.
  • Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells).
  • the nerve cell loss is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques, e.g., HPLC or FPLC) until a single compound or minimal compound mixture is demonstrated to prevent or delay the TREND-associated condition.
  • Candidate antagonists include peptide as well as non-peptide molecules (e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured).
  • Antagonists found to be effective at the level of delay of nematode nerve cell loss may be confirmed as useful in mammalian models. If desired, treatment with an antagonist of the invention may be combined with any other TREND disease therapy. Therapy
  • Compounds identified using any of the methods disclosed herein may be administered to patients with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients.
  • intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Transgenic animals of the invention include those which express a TREND-associated nucleic acid in the nervous system of the animal, thereby producing a model system for the study of TREND diseases and the screening of useful therapeutics.
  • TREND-associated nucleic acid is a Huntingtin nucleic acid (for example, a fragment encoding the first 17 amino acids of the human Huntingtin protein linked at the C-terminus to at least 119, more preferably, at least 173 glutamine residues).
  • Other nucleotides which may be expressed in these animals include, without limitation, all or a portion of the genes for SBMA, DRPLA, and the SCAS, including sufficient triplet repeat residues to confer nerve cell dysfunction. (See Gusella et al., Molecular Medicine (1997) 3:238-242; and Nasir et al., Human Molecular Genetics (1996) 5_:1431-1435). Also included are non-CAG repeat TREND nucleic acids.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene- polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for antagonists or agonists of the invention include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • TREND-associated gene may be specifically inserted within the OSM- 10 protein coding region (for example, as described herein) of the osm- 10 gene using the techniques described herein as a means to generate adult transgenic nematodes with progressive neuronal cell death.
  • the sequence generally will possess its own termination codon.
  • a polyadenylation site may be added following the coding sequence.

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Abstract

L'invention concerne des nématodes transgéniques qui constituent des systèmes modèles pour les maladies neurologiques à répétition par triplet (TRENDS).
EP98935611A 1997-07-11 1998-07-10 Modele de nematode transgenique de maladies neurologiques a repetition par triplet Withdrawn EP0996708A4 (fr)

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