WO2000021362A1 - ANIMAUX TRANSGENIQUES PrP (PROTEINE DE PRION) VIABLES ET PROCEDES D'UTILISATION - Google Patents

ANIMAUX TRANSGENIQUES PrP (PROTEINE DE PRION) VIABLES ET PROCEDES D'UTILISATION Download PDF

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WO2000021362A1
WO2000021362A1 PCT/US1999/023242 US9923242W WO0021362A1 WO 2000021362 A1 WO2000021362 A1 WO 2000021362A1 US 9923242 W US9923242 W US 9923242W WO 0021362 A1 WO0021362 A1 WO 0021362A1
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prp
animal
gene
transgenic animal
copper
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PCT/US1999/023242
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Stanley B. Prusiner
Jiri G. Safar
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The Regents Of The University Of California
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    • 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
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • 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
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • 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
    • 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
    • A01K2217/20—Animal model comprising regulated expression system
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • A01K2267/0343—Animal model for prion disease

Definitions

  • the invention relates generally to the field of non-human, transgenic animals altered with respect to the expression of the PrP gene.
  • Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not infect another (e.g., a mouse).
  • PrP Sc The infectious, disease-causing PrP isoform was designated PrP Sc molecules.
  • PrP Sc the infectious, disease-causing PrP isoform of the prion protein (PrP Sc ) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See Prusiner, S.B., "Molecular biology of prion disease," Science 252:1515-1522 (1991).
  • the most common prion diseases of animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith, J.
  • PrP c The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
  • PrP c occurred in response to the unexpected observation that the level of PrP mRNA in normal, control animals was the same as that in ill, scrapie, infected rodents (Chesebro et al, 1985, Oesch et al., 1985). PrP c was readily distinguished from PrP Sc because is was hydro lyzed under conditions that produced PrP 27-30 from PrP Sc and PrP c could be solubilized by non-denaturing detergents while PrP Sc remained insoluble (Meyer et al, 1986).
  • PrP c carries a glycosylphosphatidyl inositol (GPI) moiety provided insight into the subcellular trafficking of this protein, it did not reveal the function of PrP (Stahl et al, 1987; Taraboulos et al., 1995).
  • GPI glycosylphosphatidyl inositol
  • the high histidine content of PrP raised the possibility the immobilized metal ion affinity chromatography (IMAC) might be employed in its purification (Sulkowski, 1989).
  • IMAC immobilized metal ion affinity chromatography
  • mice with an ablated endogenous PrP gene were also found to not be susceptible to prion-mediated disorders due to the absence of the functional endogenous PrP protein. See U.S. Pat No. 5,698,763. Although these animals appeared to develop and survive like their normal counterparts, it has been found that these mice display certain dietary sensitivities that were not discovered in the initial studies of the Prnp 0/0 animals. Upon specific dietary deprivation, the Prnp 0/0 animals exhibited severe and debilitating pathological phenotypes which were not seen in similarly deprived normal mice.
  • the transgenic animal may have an endogenous gene: altered to decrease endogenous PrP expression; ablated to eliminate endogenous PrP expression; or modified to express an exogenous form of PrP.
  • the exogenous form of PrP includes a PrP gene from a genetically diverse species, a chimeric PrP gene containing sequences of the endogenous PrP gene from the PrP gene of a genetically diverse species; a mutated, deleted or otherwise altered form of the endogenous PrP, and PrP genes operably linked to an inducible promoter.
  • the transgenic animal of the invention is a rat, a hamster, or more preferably a mouse.
  • One object of the invention is to provide a transgenic, non-human animal which has its endogenous PrP gene altered to decrease or eliminate PrP expression.
  • the pathological phenotype displayed by these animals upon copper deprivation is suppressed by the administration and/or monitoring of copper to the animals, which suppresses the pathological phenotype associated with reduced PrP expression.
  • Copper is preferably maintained through the dietary administration of Cu2+ and/or monitoring of dietary Cu2+ levels.
  • the animals of this invention have an ablated endogenous PrP gene, and the transgenic animal is avian or mammalian and is provided a diet or is administered a formulation which comprises Cu2+ or provides Cu2+ to the animal upon digestion and/or administration.
  • the diet or formulation of Cu2+ is provided in an amount sufficient to suppress pathologies of mammals not provided with such.
  • One advantage of the invention is that it provides animals resistant to prion infection that remain healthy and develop normally.
  • Another object of the invention is to provide a transgenic, non-human animal which has its genome altered to 1) decrease endogenous PrP expression and 2)express an exogenous PrP gene.
  • the transgenic animal has an ablated endogenous PrP gene
  • the exogenous gene may be from a genetically diverse animal or a manipulated PrP gene such as a chimeric PrP gene comprised of codons from the host mammal and a genetically diverse mammal.
  • the exogenous gene may be from genetically similar animals.
  • the pathological phenotype displayed by these animals upon copper deprivation is suppressed by the administration and/or monitoring of copper to the animals, which suppresses the pathological phenotype associated with reduced PrP expression.
  • One advantage of the invention is that it provides PrP ablated animals that can be maintained by copper and that are susceptible to prions that normally infect genetically diverse species.
  • a feature of the invention is that the PrP gene of the transgenic animal can be altered by replacing codons with codons of a test animal at the same relative position which differ from the codons of the host animal, up to and including replacing all the differing codons wherein the codons are replaced in a manner so as to maintain the operability of the gene.
  • Yet another object of the invention is to provide a transgenic, non-human animal which has its genome altered to 1) decrease endogenous PrP expression and 2)express an exogenous PrP gene with an inducer sequence which affects the expression of the exogenous
  • the transgenic animal has an ablated endogenous PrP gene.
  • the pathological phenotype displayed by these animals upon copper deprivation is suppressed by the administration and/or monitoring of copper to the animals, which suppresses the pathological phenotype associated with reduced PrP expression.
  • Yet another object of the invention is to provide for a method of testing samples for the presence of prions. The method involves providing transgenic animals with ablated endogenous PrP genes, maintaining said animals by the administration of copper, and inoculating the animal with material suspected of containing prions.
  • An advantage of the present invention is that the transgenic animal can be used to assay for the presence of prions in a genetically diverse sample without an intervening pathological phenotype caused by decreased endogenous PrP expression.
  • transgenic animals inoculated with prions of humans can be used as test animals for testing drugs for efficacy in the treatment of humans suffering from diseases resulting from infection with prions.
  • the transgenic and hybrid animals can detect prions in a sample at very low levels, e.g., 1 part per million, and even as low as 1 part per billion.
  • the transgenic and hybrid animals provide an assay which is highly accurate, i.e., does not provide false positives and consistently determines the presence of prions.
  • Figure 1 is a bar graph illustrating ceruloplasmin levels in copper deprived mice. Ceruloplasmin activity is measured in the sera of PrnP 00 , FVB and Tg(MoPrP-A)4053/FVB mice, both on a normal and on a copper-deprived diet.
  • Figure 2 is a survival curve of neonatal mice maintained on a copper restricted diet. Percent survival is plotted as a function of the time neonatal mice were fed a copper deficient diet. PrnP 0/0 , FVB and Tg(MoPrP-A)4053/FVB neonatal pups were copper deprived beginning at four days of age.
  • Figure 3 is a bar graph showing a semi-quantitative assessment of reactive astrocytic gliosis in the cerebellar cortex of mice fed a normal diet (+Cu) and mice on a copper deficient diet (-Cu).
  • KO PrnP 00
  • WT FVB
  • Tg Tg(MoPrP-A)4053/FVB.
  • treatment used herein to generally mean obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease (e.g., a prion disease) or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease (e.g., a prion disease) or adverse effect attributable to the disease.
  • treatment covers any treatment of a disease in a mammal, particularly a cow, pig, sheep, mouse or human, and includes:
  • a disease such as a prion disease or symptoms from occurring in a subject which may be predisposed to the disease or symptom or infected with a material such as prion particles but has not yet been diagnosed as having a disease which can include the use of gene therapy;
  • a disease such as prion disease symptom, i.e., causing regression of a disease, e.g., a prion disease or prion disease symptoms.
  • a disease detrimental to early development refers to an exogenous gene which if expressed during early development (from conception to adulthood) would hinder development of the transgenic animal.
  • the invention is most useful if the exogenous gene is lethal to the animal if expressed during early development or is at least lethal to a majority of the offspring, causing 50% or more, 75% or more, 95% or more or even 100% lethality. With the invention, the gene is turned off during early development and on during adulthood.
  • Preferred genes detrimental to early development are those with an adverse effect on the nervous system, e.g., a PrP gene or ⁇ -synuclein gene.
  • isolated shall mean separated away from its natural environment.
  • An isolated protein is not necessarily separated away from all materials it is normally present with and may remain glycosylated.
  • FVB refers to a mouse strain commonly used in the production of transgenic mice.
  • PrP mouse prion protein
  • Prnp- 0,0 or Prnp-Abl refers to a transgenic animal which has its PrP gene ablated with the " 0/0 " indicating that both alleles are ablated whereas o/+ indicates only one is ablated.
  • the animal being referred to is generally a transgenic mouse which has its PrP gene ablated i.e., a PrP knockout mouse. In that the PrP gene is disrupted no mouse PrP protein is expressed.
  • CJD sporadic CJD
  • CJD Creutzfeldt- Jakob Disease
  • Iatrogenic CJD refers to disease resulting from accidental infection of people with human prions. The most noted example of such is the accidental infection of children with human prions from contaminated preparations of human growth hormone.
  • Familial CJD refers to a form of CJD which occurs rarely in families and is inevitably caused by mutations of the human prion protein gene. The disease results from an autosomal dominant disorder. Family members who inherit the mutations succumb to CJD.
  • GSS Garnier-Strassler-Scheinker Disease
  • the term “prion” shall mean an infectious particle known to cause diseases (spongiform encephalopathies) in humans and animals.
  • the term “prion” is a contraction of the words “protein” and “infection” and the particles are comprised largely if not exclusively of PrP Sc molecules encoded by a PrP gene which expresses PrP c which changes conformation to become PrP Sc . Prions are distinct from bacteria, viruses and viroids.
  • prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats.
  • BSE bovine spongiform encephalopathies
  • prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt- Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI).
  • CJD Creutzfeldt- Jakob Disease
  • GSS Gerstmann-Strassler-Scheinker Disease
  • FFI fatal familial insomnia
  • prion includes all forms of prions causing all or any of these diseases or others in any animals used ⁇ and in particular in humans and in domesticated farm animals.
  • PrP gene and "prion protein gene” are used interchangeably herein to describe genetic material which expresses proteins as shown in Figures 3-5 and polymorphisms and mutations such as those listed herein under the subheading "Pathogenic Mutations and Polymorphisms.” Unless stated otherwise the term refers to the native wild- type gene and not to an artificially altered gene.
  • the PrP gene can be from any animal including the “host” and “test” animals described herein and any and all polymorphisms and mutations thereof, it being recognized that the terms include other such PrP genes that are yet to be discovered.
  • PrP gene refers generally to any gene of any species which encodes any form of a PrP amino acid sequences including any prion protein. Some commonly known PrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA 59:9097-9101 (1992) which is incorporated herein by reference to disclose and describe such sequences.
  • standardized prion preparation "prion preparation,” “preparation” and the like are used interchangeably herein to describe a composition containing prions which composition is obtained from brain tissue of mammals which contain substantially the same genetic material as relates to PrP proteins, e.g., brain tissue from a set of mammals which exhibit signs of prion disease which mammals may comprise any of (1) a PrP chimeric transgene; (2) have an ablated endogenous PrP gene; (3) have a high copy number of PrP genes from a genetically diverse species; or (4) are hybrids with an ablated endogenous PrP gene and a PrP gene from a genetically diverse species.
  • the mammals from which standardized prion preparations are obtained exhibit clinical signs of CNS dysfunction as a result of inoculation with prions and/or due to developing the disease due to their genetically modified make up, e.g., high copy number of PrP genes.
  • the term "chimeric PrP gene” describes recombinantly constructed genes which when included in the genome of a host animal (e.g., a mouse) will render the mammal susceptible to infection from prions which naturally only infect a genetically diverse test mammal, e.g., human, bovine or ovine.
  • an artificial gene will include the codon sequence of the PrP gene of the mammal being genetically altered with one or more (but not all, and generally less than 40) codons of the natural sequence being replaced with a different codon — preferably a corresponding codon of a genetically diverse mammal (such as a human).
  • the genetically altered mammal being used to assay samples for prions which only infect the genetically diverse mammal.
  • a chimeric gene will, when inserted into the genome of a mammal of the host species, render the mammal susceptible to infection with prions which normally infect only mammals of the second species.
  • a useful chimeric gene is MHu2M which contains the starting and terminating sequence of a mouse PrP gene and a nonterminal sequence region which is replaced with a corresponding human sequence which differs from a mouse PrP gene in a manner such that the protein expressed thereby differs at nine residues. Examples of such chimeric genes can be found in U.S. Patent No. 5,565,186 which is herein incorporated by reference.
  • host animal and "host mammal” are used to describe animals which will have their genome genetically and artificially manipulated so as to include genetic material which is not naturally present within the animal.
  • host animals include mice, hamsters and rats which have their endogenous PrP gene altered by the insertion of an artificial gene or by the insertion of a native PrP gene of a genetically diverse test animal.
  • test animal and “test mammal” are used to describe the animal which is genetically diverse from the host animal in terms of differences between the PrP gene of the host animal and the PrP gene of the test animal.
  • the test animal may be any animal for which one wishes to run an assay test to determine whether a given sample contains prions with which the test animal would generally be susceptible to infection.
  • the test animal may be a human, cow, sheep, pig, horse, cat, dog or chicken, and one may wish to determine whether a particular sample includes prions which would normally only infect the test animal. This is done by including PrP gene sequences of the test animal into the host animal, inoculating the host animal with prions which would normally only infect the test animal.
  • a mouse PrP gene is genetically diverse with respect to the PrP gene of a human, cow or sheep, but is not genetically diverse with respect to the PrP gene of a hamster. In general, prions of a given animal will not infect a genetically diverse animal.
  • ablated prion protein gene means an endogenous prion protein gene which has been altered (e.g., add and/or remove nucleotides) in a manner so as to render the gene inoperative.
  • endogenous prion protein gene which has been altered (e.g., add and/or remove nucleotides) in a manner so as to render the gene inoperative.
  • nonfunctional PrP genes and methods of making such are disclosed in B ⁇ eler, H., et al "Normal development of mice lacking the neuronal cell-surface PrP protein” Nature 356, 577-582 (1992) which is incorporated herein by reference. Both alleles of the genes are disrupted.
  • hybrid animal transgenic hybrid animal and the like are used interchangeably herein to mean an animal obtained from the cross-breeding of a first animal having an ablated endogenous PrP gene with a second animal which includes either (1) a chimeric gene or artificial PrP gene or (2) a PrP gene from a genetically diverse animal.
  • a hybrid mouse is obtained by cross-breeding a mouse with an ablated mouse PrP gene with a mouse containing (1) human PrP genes (which may be present in high copy numbers) or (2) chimeric genes.
  • hybrid includes any offspring of a hybrid including inbred offspring of two hybrids provided the resulting offspring is susceptible to infection with prions with normal infect only a genetically diverse species.
  • transgenic or hybrid test animal which develops a prion disease if inoculated with prions which would normally only infect a genetically diverse test animal.
  • the terms are used to describe a transgenic or hybrid animal such as a transgenic mouse Tg(MHu2M) which, without the chimeric PrP gene, would not be susceptible to infection with a human prion (less than 20% chance of infection) but with the chimeric gene is susceptible to infection with human prions (80% to 100% chance of infection).
  • resistant to infection means the animal includes a PrP gene which renders the animal resistant to prion disease when inoculated with an amount and type of prion which would be expected to cause prion disease in the animal.
  • incubation time shall mean the time from inoculation of an animal with a prion until the time when the animal first develops detectable symptoms of disease resulting from the infection.
  • a reduced incubation time is six months or less, preferably about 75 days ⁇ 25 days or less, more preferably about 30 days ⁇ 10 days or less.
  • suppress shall mean to lessen the effects of a visible, undesirable phenotype. Although suppression may not be an complete elimination of the phenotype, it preferably alleviates the phenotype to the extent that the phenotype will not interfere with normal development, health, and/or behavior of the animal. In a preferred embodiment, suppression of a pathological phenotype results in mice in which the phenotype is undetectable.
  • BSE for bovine spongiform encephalopathy
  • CJD Creutzfeldt- Jakob Disease
  • FVB for a standard inbred strain of mice often used in the production of transgenic mice since eggs of FVB mice are relatively large and tolerate microinjection of exogenous DNA relatively well;
  • Mhu2M for a chimeric mouse/human PrP gene wherein a region of the mouse PrP gene is replaced by a corresponding human sequence which differs from mouse PrP at 9 codons;
  • Mo for mouse
  • MoPrP for a mouse PrP protein
  • MoPrP Sc for the scrapie isoform of the mouse PrP protein; p m p ⁇ / o f or a bi a ion of both alleles of an endogenous PrP protein gene, e.g., the Mo PrP gene; PrP Sc for the scrapie isoform of the PrP protein;
  • SHa for a Syrian hamster
  • SHa PrP for a Syrian hamster PrP protein
  • Tg for transgenic
  • Tg(BovPrP) for transgenic mice containing the complete cow PrP gene
  • Tg(HuPrP) for transgenic mice containing the complete human PrP gene
  • Tg(HuPrP)/Prnp 0/0 for a hybrid mouse obtained by crossing a mouse with a human PrP protein gene (HuPrP) with a mouse with both alleles of the endogenous PrP protein gene disrupted;
  • Tg(MHu2M) mice are transgenic mice of the invention which include the chimeric MHu2M gene;
  • Tg(SHa PRP) for a transgenic mouse containing the PrP gene of a Syrian hamster; Tg(SHa PrP) for transgenic mice containing the complete sheep PrP gene.
  • Tg(tetO-MoPrP) for transgenic mice containing an active portion of the tetracycline operon, the tetO-heptamer element, operably linked to the mouse PrP gene.
  • Tg(tetO-LacZ/MoPrP) for transgenic mice containing a tetO-heptamer element, and both MoPrP-A and ⁇ -galactosidase (LacZ) on either side of the tetO heptamer in opposite orientation.
  • PrP NUCLEIC ACID COMPOSITIONS The term "PrP" is used generically to designate PrP genes, e.g. homologs from rat, human, mouse, guinea pig, etc. , and their alternate forms.
  • this term encompasses different isoforms, polymorphisms, variant sequences, and mutated forms of PrP as well.
  • the term is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 1 kb beyond the coding region, but possibly further in either direction.
  • the DNA sequences encoding PrP may be cDNA or genomic DNA or a fragment thereof.
  • the gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • a genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It may further include the 3' and 5' untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' or 3' end of the transcribed region.
  • the genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence.
  • sequence of this 5' region, and further 5' upstream sequences and 3' downstream sequences, may be utilized for promoter elements, including enhancer binding sites, that provide for expression in tissues where PrP is expressed.
  • tissue specific expression is useful for determining the pattern of expression, and for providing promoters that mimic the native pattern of expression.
  • Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease.
  • mutations may be introduced into the promoter region to determine the effect of altering expression in experimentally defined systems.
  • the regulatory sequences may be used to identify cis acting sequences required for transcriptional or translational regulation of PrP expression, especially in different tissues or stages of development, and to identify cis acting sequences and trans acting factors that regulate or mediate expression.
  • Such transcription or translational control regions may be operably linked to a PrP gene in order to promote expression of wild type or altered PrP or other proteins of interest in cultured cells, or in embryonic, fetal or adult tissues, and for gene therapy.
  • the nucleic acid compositions used in the subject invention may encode all or a part of the PrP polypeptides as appropriate. Fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 18 nt, more usually at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used.
  • Homologs of cloned PrP are identified by various methods known in the art. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in lXSSC. Sequence identity may be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM saline/0.9 mM sodium citrate). By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes may be any species, e.g.
  • PrP sequence including flanking promoter regions and coding regions, may be mutated in various ways known in the art to generate targeted changes in promoter strength, sequence of the encoded protein, etc.
  • the sequence changes may be substitutions, insertions or deletions.
  • Deletions may include large changes, such as deletions of a domain or exon.
  • Other modifications of interest include epitope tagging, e.g. with the FLAG system, HA, etc.
  • fusion proteins with green fluorescent proteins (GFP) may be used.
  • Such mutated genes may be used to study structure-function relationships of apo polypeptides, or to alter properties of the proteins that affect their function or regulation.
  • Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al, 1993 Biotechniques 14:22 ; Barany, 1985 Gene 37:111-23; Colicelli et al., 1985 Mol Gen Genet 199:537-9; and Prentki et al., 1984 Gene 29:303-13. Methods for site specific mutagenesis can be found in Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, CSH Press, pp.
  • PrP genes For example, a chicken, bovine, sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al., Proc. Natl. Acad. Sci. USA 59:9097-9101 (1992). The sequence for the Syrian hamster is published in Basler et al., Cell 46:411-428 (1986). The PrP gene of sheep is published by Goldmann et al., Proc. Natl. Acad. Sci. USA 57:2476-2480 (1990). The PrP gene sequence for bovine is published in Goldmann et al, J. Gen. Virol. 72:201-204 (1991).
  • PrP gene sequence for chicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci. USA 55:7664-7668 (1991).
  • PrP gene sequence for mink is published in Kretzschmar et al., Gen. Virol. 75:2757-2761 (1992).
  • the human PrP gene sequence is published in Kretzschmar et al, DNA 5:315-324 (1986).
  • the PrP gene sequence for mouse is published in Locht et al, Proc. Natl. Acad. Sci. USA 55:6372-6376 (1986).
  • PrP gene sequence for sheep is published in Westaway et al., Genes Dev. 5:959-969 (1994).
  • PrP deletion constructs have been shown to produce smaller, more soluble PrP protein that are still capable of producing the scrapie form of the protein. These constructs may be incorporated into transgenes with altered expression.
  • a transgene is mouse PrP 106, which lacks residues 23-88 and 141-176. and has a six histidine tag in-frame bridging residues 140 and 177, is converted into a PrP Sc like molecule.
  • Both full-length and fragment transgenes may also include non-PrP sequences, such as epitope tags.
  • transgene is used herein to describe genetic material that has been or is about to be artificially inserted into the genome of a cell, particularly a mammalian cell for implantation into a living animal.
  • the transgene is used to transform a cell, meaning that a permanent or transient genetic change, preferably a permanent genetic change, is induced in a cell following incorporation of exogenous DNA.
  • a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
  • Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like.
  • transgenic mammals e.g. cows, pigs, goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.
  • Transgenic animals comprise an exogenous nucleic acid sequence present as an extrachromosomal element or stably integrated in all or a portion of its cells, especially in germ cells. Unless otherwise indicated, it will be assumed that a transgenic animal comprises stable changes to the germline sequence.
  • "chimeras” or “chimeric animals” are generated, in which only a subset of cells have the altered genome. Chimeras are primarily used for breeding purposes in order to generate the desired transgenic animal. Animals having a heterozygous alteration are generated by breeding of chimeras. Male and female heterozygotes are typically bred to generate homozygous animals. Transgenic animals fall into two groups, colloquially termed "knockouts" and
  • knockouts have a partial or complete loss of function in one or both alleles of the endogenous PrP gene.
  • Knockins have an introduced transgene with altered genetic sequence and function from the endogenous gene. The two may be combined, such that the naturally occurring gene is disabled, and an altered form introduced.
  • a knockout preferably the target gene expression is undetectable or insignificant.
  • a knock-out of a PrP gene means that function of the PrP receptor has been substantially decreased so that expression is not detectable or only present at insignificant levels.
  • transgenic knockouts have a partial or complete loss of function in one or both alleles of the endogenous PrP gene. This may be achieved by a variety of mechanisms, the preferred being homologous recombination. See e.g. U.S. Patents 5,464,764, 5,627,059 and related patents and publications to Capecchi et al., which are incorporated herein in their entirety. Other mechanisms include introduction of a disruption of the coding sequence, e.g.
  • a chromosomal deletion of all or part of the native gene may be induced, including deletions of the non-coding regions, particularly the promoter region, 3' regulatory sequences, enhancers, or deletions of gene that activate expression of PrP genes.
  • a functional knock-out may also be achieved by the introduction of an anti-sense construct that blocks expression of the native genes (for example, see Li and Cohen (1996) Cell 85:319-329).
  • “Knock-outs” also include conditional knock-outs, for example where alteration of the target gene occurs upon exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.
  • a substance that promotes target gene alteration e.g. Cre in the Cre-lox system
  • introduction of an enzyme that promotes recombination at the target gene site e.g. Cre in the Cre-lox system
  • a "knock-in" of a target gene means an alteration in a host cell genome that results in altered expression or function of the native PrP gene. Increased (including ectopic) or decreased expression may be achieved by introduction of an additional copy of the target gene, or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene. These changes may be constitutive or conditional, i.e. dependent on the presence of an activator or represser.
  • the exogenous gene is usually either from a different species than the animal host, or is otherwise altered in its coding or non-coding sequence.
  • the introduced gene may be a wild-type gene, naturally occurring polymorphism, or a genetically manipulated sequence, for example having deletions, substitutions or insertions in the coding or non-coding regions.
  • the introduced sequence may encode a PrP polypeptide, or may utilize the PrP promoter operably linked to a reporter gene.
  • the introduced gene is a coding sequence, it is usually operably linked to a promoter, which may be constitutive or inducible, and other regulatory sequences required for expression in the host animal.
  • operably linked is meant that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules, e.g. transcriptional activator proteins, are bound to the regulatory sequence(s).
  • constructs of interest include anti-sense PrP, which will block native PrP expression, expression of dominant negative PrP mutations, and over-expression of a PrP gene.
  • a detectable marker such as lac Zmay be introduced into the locus, where upregulation of expression will result in an easily detected change in phenotype.
  • Constructs utilizing the PrP promoter region, in combination with a reporter gene or with the coding region are also of interest.
  • a series of small deletions and/or substitutions may be made in the PrP gene to determine the role of different exons in DNA binding, transcriptional regulation, etc.
  • the exogenous PrP gene may be any mammalian PrP gene, and may be a wild-type gene, a naturally occurring polymorphism, or a genetically manipulated sequence, for example having deletions, substitutions or insertions in the coding or non-coding regions.
  • the introduced sequence may encode a PrP polypeptide, or may utilize the PrP promoter operably linked to a reporter gene. Where the introduced gene is a coding sequence, it is usually operably linked to a promoter, which may be constitutive or inducible, and other regulatory sequences required for expression in the host animal.
  • operably linked is meant that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules, e.g. transcriptional activator proteins, are bound to the regulatory sequence(s).
  • constructs of interest include, but are not limited to, anti-sense PrP, which will block native PrP expression, expression of dominant negative PrP mutations, and over-expression of a PrP gene.
  • a detectable marker such as lac Z may be introduced into the locus, where upregulation of expression will result in an easily detected change in phenotype.
  • Constructs utilizing the PrP promoter region, in combination with a reporter gene or with the coding region are also of interest.
  • DNA constructs for homologous recombination will comprise at least a portion of the PrP gene with the desired genetic modification, and will include regions of homology to the target locus.
  • DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included.
  • Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990) Methods in Enzymology 185:527-537.
  • ES embryonic stem
  • an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g.
  • ES cells When ES cells have been transformed, they may be used to produce transgenic animals. See U.S. Patents 5,387,742, 4,736,866 and 5,565,186 for methods of making transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection.
  • appropriate growth factors such as leukemia inhibiting factor (LIF).
  • LIF leukemia inhibiting factor
  • Blastocysts are obtained from 4 to 6 week old superovulated females.
  • the ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct.
  • chimeric progeny can be readily detected.
  • the chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allergenic or congenic grafts or transplants, or in in vitro culture.
  • a number of different transgenic PrP animals can be made in the practice of the invention. The production and use of certain PrP transgenic animals are described in USSN 08/692,892 and 09/052,963 which are incorporated herein by reference. Crossed With MoPrP Gene Ablated Mice
  • FVB mice expressing human PrP genes have been constructed using the cos.SHaTet cosmid expression vector derived from the Syrian hamster (SHa).
  • SHa Syrian hamster
  • the FVB strain of mice contain and express the normal complement of MoPrP genes and so one method for introducing the HuPrP transgene array into a background in which MoPrP expression is ablated is by genetic crosses between the transgenic FVB-derived line and a second line of transgenic mice in which both MoPrP genes were disrupted. Mice homozygous for the disrupted Prnp genes were created.
  • mice were created by a process known as homologous recombination (Thomas and Capecchi, Cell 51:503-512, 1987) in which a selectable disrupted MoPrP gene was introduced into embryonic stem (ES) cells from SV129 mice.
  • ES embryonic stem
  • Blastocysts of C57BL/6J mice were injected with SV129 ES cells in which one copy of the MoPrP gene had been disrupted thus generating a chimeric mouse with one disrupted allele. That mouse was mated with a C57BL mouse and the offspring crossed to each other to produce null animals in which both copies of the MoPrP gene were disrupted, referred to as Prnp 0/0 mice.
  • Prnp 0/0 mice were repeatedly crossed onto the FVB background.
  • FVB-derived transgenic mouse lines Tg(HuPrP)FVB/152 and Tg(HuPrP)FVB/440 were crossed with Prnp 0/0 mice. Backcrossing these mice produced animals in which the only PrP c molecules that were synthesized were those encoded by the transgene.
  • the second method for producing transgenic mice in which the only PrP c molecules synthesized are encoded by the HuPrP transgene is by directly microinjecting DNA from a vector capable of directing expression of HuPrP.
  • Derivatives of the cos.SHaTet cosmid expression vector containing the HuPrP open reading frame were used — (other expression systems could be used including a cosmid consisting of the cognate HuPrP gene or other vectors capable of appropriate expression of HuPrP in transgenic mice).
  • Using embryos from the originally created C57BL-derived Prnp 0/0 mice we encountered great difficulty in producing transgenic mice by this method because of the poor survival rates of micro injected embryos.
  • Prnp 00 mice were subsequently repeatedly crossed onto the FVB background to produce mice which were genetically -95% FVB but which were also homozygous for the gene ablation.
  • Prnp 0/0 mice we now have very high rates of production of transgenic mice by this method.
  • mice are mated to transgenic mice expressing high levels of the homologous protein such as HuPrP or MHu2MPrP.
  • the mice will express the highest levels of the foreign PrP of interest and possess the shortest incubation times.
  • mice with -50 copies of the MoPrP transgene have incubation times of -60 days after inoculation with - 10 6 ID 50 units if the endogenous MoPrP genes are ablated; in contrast, incubation times of -48 days were found if the endogenous MoPrP genes are left intact (Table 8).
  • the fertilized eggs from these mice with gene replacements can be microinjected with the DNA encoding either the same PrP gene as that replaced or a related gene.
  • a different approach to eliminating the inhibitory effects of MoPrP would be to create new lines of transgenic mice in which the endogenous MoPrP genes were replaced with MoPrP genes or HuPrP genes operably linked to an inducible promoter.
  • the expression of the exogenous PrP gene would be temporally and/or qualitatively using an inducible system such as that disclosed in Shockett, PNAS 43:5173-5176 (1996), which is incorporated by reference herein.
  • the pioneering tet-regulated gene expression system involved a constitutive expression of the tet transactivator protein (tTA) with the cytomegalovirus (CMV) immediate early (IE) promoter/enhancer.
  • tTA tet transactivator protein
  • CMS cytomegalovirus
  • IE immediate early
  • a modified system has also been developed using a reverse transactivator (rtTA) that binds tetO efficiently only in the presence of the tet derivatives doxycycline or anhydrotetracycline.
  • rtTA reverse transactivator
  • binds tetO efficiently only in the presence of the tet derivatives doxycycline or anhydrotetracycline may be used in the present invention without departing from the spirit of the disclosure, as will be obvious to those skilled in the art.
  • systems such as ecdysome inducible systems can be used instead of the tetracycline inducible system.
  • the transactivator of the inducible system may be introduced via the CMV promoter, viral vectors driven by either the SV40 promoter, by glial-cell specific promoters, or by the autonomous parvo virus LuIII.
  • FVB mice expressing human, chimeric Hu/Mo and mutant PrP genes were constructed using the cos.SHaTet cosmid expression vector derived from the Syrian hamster (SHa) (Scott et al., 1992). Table 1 below shows the designation of the mouse line, the expressed PrP c molecules and the approximate level of transgenic expression. Also indicated are those mouse lines that were crossed with Prnp 0/0 mice in which the mouse PrP gene had been disrupted by homologous recombination (B ⁇ eler et al., 1992). Backcrossing these mice produced animals in those encoded by the transgene. While SV129ES cells were used to generate a chimeric mouse with a disrupted PrP allele, that mouse was mated with a C57BL mouse and the offspring crossed to each other to produce null animals.
  • COPPER ADMINISTRATION Copper may be administered to the animals of the invention using any convenient means capable of administering the copper in the desired quantity.
  • the agent can be incorporated into a variety of formulations for administration. More particularly, the agents of the present invention can be formulated into feed and/ or compositions by combination with appropriate materials. Formulations which contain copper and/or copper derivatives which result in copper which can be absorbed into a living organism can be injected or administered by any other means known to those skilled in the art.
  • the copper is added in a metabolically accessible form to the feed, for example as a chloride, sulfate and/or organic chelates, and the copper is ingested by the subject to be treated.
  • Dietary ingestion of the copper is the method of administration of the preferred embodiment of the instant invention.
  • pharmaceutically acceptable carriers or diluents may be added, and the formulation may be prepared in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc., administration.
  • the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxy methylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxy methylcellulose
  • lubricants such as talc or magnesium stearate
  • the agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • solubilizers isotonic agents
  • suspending agents emulsifying agents
  • stabilizers and preservatives emulsifying agents
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifiuoromethane, propane, nitrogen and the like.
  • the copper can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • the copper used of the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • Standardization prion preparations are produced for use in assays so as to improve the reliability of the assy. Preparation can be obtained from the animals of the invention and such preparations are produced as described in USSN 08/521,992 which is herein incorporated by reference.
  • mice and hamsters are preferred host animals.
  • mice and hamsters are preferred host animals.
  • Other possible host animals include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats), Oryctolagus (e.g. rabbits), and Mesocricetus (e.g. hamsters) and Cavia (e.g., guinea pigs).
  • Mus e.g. mice
  • Rattus e.g. rats
  • Oryctolagus e.g. rabbits
  • Mesocricetus e.g. hamsters
  • Cavia e.g., guinea pigs
  • the host PrP gene can be changed to include codons from genetically diverse PrP genes from test animals belonging to a genus selected from Bos, Ovis, Sus and Homo.
  • a mouse host PrP gene is changed to include codons from a human, cow or sheep PrP gene, with human being most preferred.
  • Humans are preferred because an important object of the invention is to use the animal to test a sample of material to determine if that material has prions which will infect a human and cause a human to develop a CNS disease such as CJD.
  • Preferred transgenic animals are disclosed in U.S. Patent 5,565,186 issued October 15, 1996 and WO 97/04814 published February 13, 1997 which are incorporated herein by reference to disclose transgenic animals and methods of making and using such.
  • the genetic material which makes up the PrP gene is known for a number of different species of animals (see Gabriel et al., Proc. Natl. Acad. Sci. USA 59:9097-9101 (1992)). Further, there is considerable homology between the PrP genes in different mammals. For example, see the amino acid sequence of mouse PrP compared to human, cow and sheep PrP in Figures 3, 4 and 5 wherein only the differences are shown. Although there is considerable genetic homology with respect to PrP genes, the differences are significant in some instances. More specifically, due to small differences in the protein encoded by the PrP gene of different mammals, a prion which will infect one mammal (e.g. a human) will not normally infect a different mammal (e.g. a mouse).
  • mice Due to this "species barrier,” it is not generally possible to use normal animals, (i.e., animal which have not had their genetic material related to prions manipulated) such as mice to determine whether a particular sample contains prions which would normally infect a different species of animal such as a human.
  • a pressure-sensitive measurement system may be employed to detect and record an inoculated animal's physiological changes, and specifically its motor skills.
  • a pressure sensitive system that would be effective in monitoring changes in walking is a platform with a piezoelectric ground reaction force (GRF) measurement system such as that used in the Gaitway Instrumented Treadmill (Kistler Biomechanics, Winterthut, Switzerland).
  • GRF piezoelectric ground reaction force
  • Such a system has the ability to measure vertical ground reaction force and center of pressure for complete, multiple foot strikes.
  • the system also has an integrated software system that can distinguish between left and right strikes and measure vertical force, center of pressure, and temporal gait parameters.
  • a database can keep track of trials by subject name, identification number, or other user-specified classification. Multiple trials can be overlaid on a single graph, and the progression of a single subject examined over time.
  • the time base can be varied to view data in absolute time, relative time, percent contact, percent step, and percent gait cycle.
  • Another method of evaluating motor coordination is a rotorod test (Sakaguchi et al.). Animals are placed on a rotating rod, whose rotating frequency is steadily increased from 10 rpm until the animals falls from it. The animal is subjected to ten trials and its best score recorded as its score on that given day. This test would offer a quantitative measurement of motor skills over the progression of prion disease and permit the early detection of motoric deficiencies.
  • Transgenic animals of the invention can be used to evaluate the efficacy of drugs.
  • the exogenous gene is turned on in two mice and the drug is administered to one but not the other mouse. The effect of the drug on inhibiting the progress of disease as compared to the mouse with no drug is then determined.
  • the exogenous gene is turned on and left on until symptoms develop in two mice. The gene is then turned off in both mice and drug administered to one mouse but not the other. Measurements are then taken over time for both mice to determine the rate at which harmful protein is cleared from the system of each mouse. The drug is determined efficacious if it increases the rate at which the harmful protein is cleared from the system as compared to the rate it is cleared from the system of the mouse with no drug.
  • Tg(MoP ⁇ -A) 4053/FVB were obtained by microinjection of one cell zygotes collected from FVB mice (Telling et al., 1996).
  • the transgenic line was maintained by breeding the transgene-positive founder with wild-type FVB animals (Charles River, Wilmington -Ma).
  • the PrP deficient animals used were obtained by backcrossing hemizygous Prnp + 0 animals (B ⁇ eler et al., 1992, Prusiner et al., 1993b) with FVB animals for 10 generations before interbreeding to homozygosity. Breeding and screening of transgenic (Tg) mice were performed as previously described (Prusiner et al, 1993b; Scott et al., 1989).
  • Wild type and transgenic mice were mated to establish a breeding colony. Four days after parturition, the mouse dams with pups were divided into two dietary treatments, -Cu and +Cu .
  • The-Cu group was fed a copper depleted diet (TD 80388; Teklad, San Diego, CA) and drank purified deionized water (Millipore, Bedford, MA). This diet contained less than 0.45 mg of copper per kilogram of pellets.
  • Mice in the +Cu group were fed a diet containing 19.4 mg/kg of copper, and drank normal drinking water ad libitum. Mice were housed in polycarbonate disposable cages with stainless steel frame and a cardboard next for pups.
  • mice were weaned at 4 weeks of age, their genotype determined, and placed five each in disposable polycarbonate cages with elevated stainless steel frame. The mice were checked for clinical symptoms, weighed daily and continued to receive the assigned diet for an additional two weeks. The experiment was terminated after 42 days. The animals were euthanized in terminal stages of disease or after 42 days. Mice euthanized in Halothan or CO 2 were decapitated and trunk bleed into 1.5 ml Eppendorf tubes. Blood was kept at room temp for 30 minutes, clots removed, and spun at 1000 ⁇ ra for 10 minutes. Collected serum was stored at -20 °C for the ceruplasmin assay and copper measurements. Brain, liver, and kidneys were removed, immediately frozen, and stored at -70 °C.
  • kidney copper levels in all three lines fed a copper restricted diet were about 10% of those found in controls when the mice were sacrificed at 42 days after dietary copper deprivation was initiated. In liver and kidney, all the mice showed substantially reduced copper levels after dietary copper deprivation except for the Prnp 0/0 mice. Although kidney copper levels in p m p ⁇ / o m j ce were approximately 50% of those in FVB mice fed a normal diet, there was only a modest reduction in kidney copper when the Prnp 00 were fed a copper restricited diet. Curiously the kidney copper levels in the Tg(MoPrP-A)4053/FVB mice fed a normal diet were similar to those in Prnp 00 mice. In contrast to Prnp 00 mice, kidney copper in
  • Tg(MoPrP-A)4053/FVB mice after dietary copper deprivation was substantially reduced.
  • Serum ceruloplasmin was determined spectrophotometricly by oxidation of p- phenylenediamine as described (Rice et al, Anal. Biochem. 3:452-456 (1962 ). Briefly, 20 ⁇ l or sera was mixed with 200 ⁇ l of 0.1% (w/v) p-phenylenediamine (PPD) solution in 1.2 M acetate buffer, containing 40 ⁇ M of EDT A. The mixture was incubated for 15 min at 37°C and reaction stopped by 1 ml of 1% (w/v) NaN 3 . The absorbance was read at 540 nm and the ceruloplasmin activity was calculated from a standard calibration curve.
  • PPD p-phenylenediamine
  • mice starved for copper exhibited diminished liver weights at autopsy.
  • the livers of Prnp 0/0 mice on a copper restricted diet were 64% as large as those of control Prnp 0/0 mice fed a normal diet as shown in Table 3 below.
  • livers of FV13 and Tg(MoPrP-A)4053/FVB mice on a copper restiricted diet were 71% and 48% as large at the time of sacrifice as those of control mice fed a normal diet, respectively.
  • no significant decrease in brain or kidney weights were found in mice fed a copper restricted diet.
  • Copper measurements were performed in a 2380 Perkin Elmer flameless graphite furnace atomic abso ⁇ tion spectrophotometer at a wavelength of 324.7 nrn after rapid atomization at 2300*C in a graphite tube cuvette HGA-70 (Perkin Elmer). The absolute amount of copper was calculated from standard calibration curve after subtracting blanks. The copper concentration for each organ are expressed in ⁇ g/g of wet tissue weight.
  • mice After a series of studies with weanling mice in which we were unable to achieve severe copper deprivation as noted above, we implemented a protocol using neonates. At four days of age, mothers and offspring were placed on a copper deficient diet. The three different lines of mice Prnp 0/0 , FVB, and Tg(MoPrP-A)4053/FVB described above were employed in these studies. After 42 days, the experiment was terminated because so many of copper-deprived animals that remained alive appeared very ill. Mice sacrificed on the terminal day of the experiment were 46 days of age.
  • the Prnp 00 neonates began to exhibit signs of CNS dysfunction.
  • 50% of the Prnp 00 mice displayed ataxia, tremor and paresis ( Figure 2).
  • These copper deprived Prnp 0/0 mice exhibited a yellow tint in their fur, had a scuffy appearance due to their failure to groom themselves and appeared emaciated.
  • the copper deficient Prnp 0/0 mice had substantially lower body weights.
  • mice deprived of dietary Cu2+ exhibited widespread astrocytic gliosis in the brainstem and cerebellum with few pathologic changes seen in the cerebrum.
  • none to the control mice fed a normal diet and sacrificed at 46 days of age showed any neuropathological changes.
  • the main neuropathological feature was reactive astrocytic gliosis without obvious nerve cell loss, nerve cell degeneration or neuronal dystrophy. Reactive astrocytic gliosis was most obvious in all copper deficient animals in the cerebellar cortex and white matter.
  • the degree of reactive astrocytic gliosis in each animal was estimated by a semiquantitative method which assesses the area of the cerebellar cortex occupied by reactive astrocytes and their processes as revealed by GFAP immunohistochemistry (Figure 3).
  • the control group of five brains consisted of one Prnp 00 , two FVB mice and two Tg(MoPrPA)4053/FVB mice all fed a normal diet.
  • the copper deprived mice, all of which had signs of neurologic dysfunction, consisted of four Prnp 0/0 mice, eight FVB mice and 16 Tg(MoPrP-A)4053/FVB mice. Each group of copper deficient mice showed statistically significant differences in the proportion of cerebellar cortex exhibiting GFAP immunoreactivity.
  • Prnp 0/0 , FVB, and Tg(MoPrP-A)4053/FVB mice were maintained on a normal, copper-sufficient diet, or fed a copper deficient diet (TD 80388) and examined for potential phenotypes.
  • the strains of mice used were produced as per Example 1. Greater than 90% of Prnp 0/0 , FVB, and Tg(MoPrP-A)4053/FVB mice maintained on a normal, copper-sufficient diet developed normally and remained well for more than 300 days. The Prnp 0/0 mice were observed for more than 760 days at which time less than 10% had developed.
  • Prnp 0/0 Mice fed a copper deficient diet showed a sha ⁇ ly declining survival rate, with less than half of the animals surviving beyond 30 days from initiation of copper deprivation.
  • Prnp 0/0 mice only 5% of the FVB and Tg(MoPrP-A)4053/FVB mice fed a copper deficient diet for 30 days displayed signs of neurologic dysfunction ( Figure 2).
  • sfter 42 days on a copper deficient diet 28% of the FVB mice and 27% of the Tg(MoPrP-A)4053/FVB mice had been sacrificed after becoming severely emaciated compared to 68% of the Prnp 0/0 mice.

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Abstract

La présente invention porte sur des animaux transgéniques présentant: 1) un génome modifié artificiellement de façon à réduire ou éliminer l'activité endogène de PrP; et 2) une bonne santé et un développement normal entretenus par l'administration de cuivre. L'animal transgénique peut avoir un gène endogène modifié qui réduit l'expression endogène de PrP; un gène qui a été retiré de façon à éliminer l'expression endogène de PrP; un gène modifié pour exprimer une forme exogène de PrP. La forme exogène de PrP comprend un gène PrP provenant d'une espèce génétiquement différente, un gène chimère PrP contenant des séquences du gène PrP endogène provenant du gène PrP d'une espèce génétiquement différente; une forme mutée, éliminée ou sinon modifiée des gènes PrP endogènes et PrP liés de manière fonctionnelle à un promoteur inductible. La souris transgénique de cette invention est utilisée dans le dosage biologique de fait de sa sensibilité aux infections par les prions, cette sensibilité étant réduite par l'administration de quantités régulées de cuivre. Les animaux de ferme transgéniques (tels que les vaches) qui sont utilisés à des fins ordinaires ont l'avantage de ne pas être sensibles aux prions qui infectent normalement les animaux de cette espèce (telle que BoPrPSc).
PCT/US1999/023242 1998-10-09 1999-10-05 ANIMAUX TRANSGENIQUES PrP (PROTEINE DE PRION) VIABLES ET PROCEDES D'UTILISATION WO2000021362A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073107A1 (fr) * 2000-03-24 2001-10-04 University Of Massachusetts, A Public Institution Of The Commonwealt Of Massachusettes, As Represented By Its Amherst Campus Ongules transgeniques depourvus de prion
WO2004047530A1 (fr) * 2002-11-27 2004-06-10 Medical Research Council Systeme de modele de prions
JP2008500849A (ja) * 2004-05-24 2008-01-17 サーントゥル ナシオナル ドゥ ラ ルシェルシュ シャーンティフィク プリオン汚染除去のための製品および方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997004814A1 (fr) * 1995-07-31 1997-02-13 The Regents Of The University Of California Detection de prions dans un echantillon, preparation de prions et animal transgenique utilises a cette fin
US5698763A (en) * 1991-11-14 1997-12-16 Weissmann; Charles Transgenic animals lacking prion proteins
WO1998028971A2 (fr) * 1997-01-03 1998-07-09 University Technology Corporation Toxicite de la beta-amyloide
WO1999015640A1 (fr) * 1997-09-22 1999-04-01 The Regents Of The University Of California Detection des prions de vache, de mouton, et de cochon dans un echantillon, et animaux transgeniques utilises a cette fin

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5698763A (en) * 1991-11-14 1997-12-16 Weissmann; Charles Transgenic animals lacking prion proteins
WO1997004814A1 (fr) * 1995-07-31 1997-02-13 The Regents Of The University Of California Detection de prions dans un echantillon, preparation de prions et animal transgenique utilises a cette fin
WO1998028971A2 (fr) * 1997-01-03 1998-07-09 University Technology Corporation Toxicite de la beta-amyloide
WO1999015640A1 (fr) * 1997-09-22 1999-04-01 The Regents Of The University Of California Detection des prions de vache, de mouton, et de cochon dans un echantillon, et animaux transgeniques utilises a cette fin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BROWN D R, ET AL.: "PRION PROTEIN-DEFICIENT CELLS SHOW ALTERED RESPONSE TO OXIDATIVE STRESS DUE TO DECREASED SOD-1 ACTIVITY", EXPERIMENTAL NEUROLOGY, ACADEMIC PRESS, NEW YORK, NY, US, vol. 146, 1 January 1997 (1997-01-01), US, pages 104 - 112, XP002901003, ISSN: 0014-4886, DOI: 10.1006/exnr.1997.6505 *
PRUSINER S B, SCOTT M R: "GENETICS OF PRIONS", ANNUAL REVIEW OF GENETICS., ANNUAL REVIEWS INC., PALO ALTO, CA, US, vol. 31, 1 January 1997 (1997-01-01), US, pages 139 - 175, XP002901001, ISSN: 0066-4197, DOI: 10.1146/annurev.genet.31.1.139 *
SHOCKETT P E, SCHATZ D G: "DIVERSE STRATEGIES FOR TETRACYCLINE-REGULATED INDUCIBLE GENE EXPRESSION", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 93, 1 May 1996 (1996-05-01), US, pages 5173 - 5176, XP000606286, ISSN: 0027-8424, DOI: doi:10.1073/pnas.93.11.5173 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073107A1 (fr) * 2000-03-24 2001-10-04 University Of Massachusetts, A Public Institution Of The Commonwealt Of Massachusettes, As Represented By Its Amherst Campus Ongules transgeniques depourvus de prion
WO2004047530A1 (fr) * 2002-11-27 2004-06-10 Medical Research Council Systeme de modele de prions
JP2008500849A (ja) * 2004-05-24 2008-01-17 サーントゥル ナシオナル ドゥ ラ ルシェルシュ シャーンティフィク プリオン汚染除去のための製品および方法

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