WO2004047530A1

WO2004047530A1 - Transgenic model expressing a prp gene homozygous for m129

Info

Publication number: WO2004047530A1
Application number: PCT/GB2003/005162
Authority: WO
Inventors: John Collinge; Emmanuel Asante
Original assignee: Medical Research Council
Priority date: 2002-11-27
Filing date: 2003-11-27
Publication date: 2004-06-10
Also published as: AU2003285535A1; GB0227651D0

Abstract

The invention relates to a transgenic non-human animal expressing, as a result of transgene expression, a PrP gene homozygous for M129, wherein contacting said transgenic non-human animal with a sample containing prions causes prion infection that is characteristic of human prion infection. The invention also relates to uses and applications of said model, processes involving it and agents identified through it.

Description

TRANSGENIC MODEL EXPRESSING A PRP GENE HOMOZYGOUS FOR M129

FIELD OF INVENTION

The present invention relates to transgenic non-human animals.

In particular, the present invention relates to transgenic non-human animals that express, as a result of transgene expression, a PrP gene homozygous for Ml 29, wherein contacting said transgenic non-human animals with a sample containing prions causes prion infection that is characteristic of human prion infection.

Moreover, the present invention also relates to methods, processes, pharmaceutical compositions, agents and uses relating to the transgenic non-human animals of the present invention.

BACKGROUND TO THE INVENTION

By way of background information, a prion protein (PrP) is a transmissable particle devoid of nucleic acid. The PrP gene encodes prion proteins. The most notable prion diseases are Bovine Spongiform Encephalopathy (BSE), Scrapie of Sheep and Creutzfeldt- Jakob Disease (CJD) of humans. The most common manifestation of CJD is sporadic CJD (sCJD) which occurs spontaneously in individuals. Iatrogenic CJD (iCJD) is a disease that results from accidental infection. Familial CJD (fCJD) is a form of CJD that occurs rarely in families and is caused by mutations of the human PrP gene. 'New variant' CJD (vCJD) of humans is a distinct strain type of CJD that is associated with a pattern of PrP glycoforms that are different from those found for other types of CJD. It has been suggested that BSE may have passed from cattle resulting in vCJD in humans.

Prion diseases are associated with accumulation of a disease-associated isoform of host-encoded cellular prion protein (PrP^c), designated PrPSc. PrPSc is thought to comprise an aggregated form of a conformational isomer of PrP . According to the protein-only hypothesis, infectious prions are composed principally, if not entirely, of an abnormal isoform of PrP. Distinctive isolates or strains of prions can be propagated in the same type of host and may be encoded by differences in PrPSc conformation (Bessen and Marsh, 1992, 1994; Collinge et al, 1996b; Telling et al, 1996) and glycosylation (Collinge et al, 1996b). Variant CJD (vCJD), recognised in 1996, is thought to be caused by exposure to BSE-like prions (Collinge et al., 1996b; Lasmezas et al., 1996; Bruce et al, 1997; Hill et al., 1997).

The demonstration that vCJD is caused by the same prion strain that causes bovine spongiform encephalopathy, has led to concerns about the possibility of a human epidemic. Although a limited number of cases of vCJD have been reported to date, it is likely that hundreds of thousands of infected cattle entered the human food chain in the late 1980s and early 1990s, and the average incubation period of vCJD is unknown.

The study of disease often requires suitable animal models which can faithfully replicate the disease conditions as seen in humans. The animals models may then be used to develop therapies.

Transgenesis is a well established technique for the introduction of DNA sequences into the mammalian genome, for example mice and other species. The methods are well known in the art and typically involve the microinjection of cloned DNA fragments into the male pro-nuclei of eggs isolated from superovulated females. Such eggs are transferred into the oviduct of pseudopregnant females (obtained by mating with vasectomized males) and carried to term. DNA extracted from tail clippings obtained from the progeny may be examined for the presence of specific transgene DNA. Depending on the integrity and stability of the DNA sequence, the number of integration sites and their location in the host genome, transgenes may become stably integrated in the host genome and transmitted to subsequent progeny.

In one animal model for prion infection that has been described, mice which express human PrP V129 (129VV Tgl52 mice), are highly susceptible to infection with human prions from patients with sporadic and iatrogenic forms of CJD, regardless of patient genotype at polymorphic codon 129 (Collinge et al., 1995; Hill et al, 1997). These mice were much less susceptible to prions from patients with vCJD. There is a continuing need in the art for new tools to study human prion disease and for new ways and means to treat and prevent this disease.

SUMMARY OF THE INVENTION

The present invention is based upon the surprising finding that transgenic non-human animals, for example, mice, expressing human PrP methionine 129, inoculated with prions develop prion infection that is characteristic of human prion infection, for example, vCJD.

Even more surprisingly, BSE prion inoculation of the transgenic non-human animals also resulted in the phenotypic characteristics of human prion infection — such as type 2 and type 4 human prion infection. In contrast to BSE transmission to 129VV Tgl52 mice where PrPSc was not detected in the brain (Hill et al., 1997), PrPSc was readily detectable in the brains of the transgenic non-human animals of the present invention.

Advantageously, the transgenic non-human animals according to the present invention may be used as in vivo experimental models to investigate and/or design therapies or therapeutic agents to modulate human prion infection. The models may be used to investigate the effect of various tools/lead compounds on a variety of parameters, which are implicated in the development of, or treatment of prion infection.

In a first aspect, the present invention relates to a transgenic non-human animal expressing, as a result of transgene expression, a PrP gene homozygous for M129, wherein contacting said transgenic non-human animal with a sample containing one or more prions causes prion infection that is characteristic of human prion infection.

Contacting the transgenic animals with prions that do not normally cause prion disease in humans, for example, prions that cause BSE, surprisingly causes the transgenic animals to develop a pathological (eg. neuropathological) and/or biochemical and/or molecular phenotype characteristic of human prion infection.

Thus, in a second aspect, the present invention relates to a method for identifying one or more agents capable of modulating human prion infection, comprising the steps of: (a) contacting a transgenic non-human animal expressing, as a result of transgene expression, a PrP gene homozygous for Ml 29, with a sample comprising one or more prions; (b) contacting the transgenic non-human animal with one or more agents; (c) determining the effect of the agent on prion infection; and (d) selecting one or more agents which are capable of modulating prion infection.

The agents identified in the method according to the second aspect of the present invention may be prepared in various ways.

Accordingly, in a third aspect, the present invention relates to a process comprising the steps of: (i) performing the assay method according to the second aspect of the present invention; (ii) identifying an agent capable of modulating human prion infection; and (iii) preparing a quantity of that agent.

In a fourth aspect, the present invention relates to a process comprising the steps of: (i) performing the assay according to the second aspect of the present invention; (ii) identifying an agent capable of modulating human prion infection; (iii) preparing a quantity of that agent; and (iv) preparing a pharmaceutical composition comprising that agent.

In a fifth aspect, the present invention relates to a process comprising the steps of: (i) performing the assay according to the second aspect of the present invention; (ii) identifying an agent capable of modulating prion infection; (iii) modifying said agent; and (iv) preparing a pharmaceutical composition comprising said modified agent.

Advantageously, the agents of the present invention may be formulated into a pharmaceutical composition for the treatment of prion infection.

Thus, in a sixth aspect, the present invention relates to a pharmaceutical composition comprising an agent identified by the assay method according to the second aspect of the present invention or the process according to the third, fourth of fifth aspects of the present invention admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant and/or combinations thereof. In a seventh aspect, the present invention relates to a process of preparing a pharmaceutical composition comprising admixing an agent identified by the assay method according to the second aspect of the present invention or the process according to the third, fourth of fifth aspects of the present invention with a pharmaceutically acceptable diluent, carrier, excipient or adjuvant and/or combinations thereof.

In an eighth aspect, the present invention relates to a method of treating human prion infection which method comprises administering to an individual an effective amount of a pharmaceutical composition comprising an agent identified by the assay method according to the second aspect of the present invention or the process according to the third, fourth of fifth aspects of the present invention, wherein the agent is capable of modulating prion infection and wherein said composition is optionally admixed with a pharmaceutically acceptable carrier, diluent excipient or adjuvant and/or combinations thereof.

In a ninth aspect, the present invention relates to an agent identifiable, preferably identified by the assay method according to the second aspect of the present invention.

In a tenth aspect, the present invention relates to an agent identifiable preferably identified by the assay method according to the second aspect of the present invention for use in the treatment and/or prevention of disease.

In an eleventh aspect, the present invention relates to the use of an agent identifiable, preferably identified by the assay method according to the second aspect of the present invention in the manufacture of a composition for the treatment and/or prevention of human prion infection.

In a twelfth aspect, the present invention relates to the use of a transgenic non-human animal according to the first aspect of the present invention as a model for human prion infection. Preferably, the PrP gene homozygous for M129 is a human PrP gene.

Preferably, the prions cause prion infection in humans or bovines. Preferably, the prions cause sCJD, vCJD or BSE in their natural hosts.

Preferably, the prions have aPENP129MM genotype.

Preferably, the human prion infection is characteristic of vCJD.

Preferably, the transgenic non-human animal is a mammal. More preferably, the mammal is a mouse.

Preferably, one or more agents are formulated into one or more compositions for use in medicine.

DESCRIPTION OF THE FIGURES

Figure 1

Immunohistochemistry of cerebral cortex and hippocampal regions of transgenic mouse brain showing abnormal PrP immunoreactivity, including PrP-positive florid plaques (enlarged in insets). (A) vCJD inoculated 129MM Tg35 mouse. (B) BSE- inoculated 129MM Tg35 mouse. (C) vCJD inoculated 129MM Tg45 mouse. (D) BSE-inoculated 129MM Tg45 mouse. (E-G) Histological analysis showing the thalamus of a BSE-inoculated 129MM Tg35 mouse propagating type 2 human PrPSc with widespread vacuolation (E; H&E), extensive gliosis (F; GFAP), but no specific PrP immunoreactive deposits (G; ICSM 35). Scale bar: (A-D) = 100 mm; (E), (F) and (G) = 50 mm.

Figure 2

Western blots of proteinase K (PK)-treated brain homogenates from transgenic mice, human cases of variant and sporadic CJD, and lines of wild-type mice. (A) Lane 1, vCJD; lane 2, vCJD-inoculated 129MM Tg35 mouse. (B) Lane 1, vCJD-inoculated 129MM Tg35 mouse; lane 2, BSEinoculated 129MM Tg35 mouse propagating type 2 PrPSc; lane 3, vCJD. (C) Lanes 1 and 2, BSE-inoculated 129MM Tg35 mouse propagating either type 2 PrPSc (lane 1) or type 4 PrPSc (lane 2). (D) Lane 1, BSE- inoculated 129MM Tg35 mouse propagating type 2 PrPSc; lane 2, human sporadic CJD type 2 PrPSc (PRNP genotype 129MM). (E) Lanes 1-3, human sporadic CJD type 2 PrPSc (PRNP genotype 129MM); lanes 4-6, BSE-inoculated 129MM Tg35 mouse propagating type 2 PrPSc. Samples were PK digested in the absence (lanes 1, 3, 4 and 6) or presence (lanes 2 and 5) of 25 mM EDTA. *Following proteolysis, samples in lanes 3 and 6 were boiled in SDS sample buffer and subsequently adjusted to 25 mM EDTA before electrophoresis. (F) Transmission of vCJD and BSE to 129MM Tg45 mice. Lane 1, vCJD; lane 2, vCJD-inoculated 129MM Tg45 mouse; lane 3, BSEinoculated 129MM Tg45 mouse. (G) Primary transmission of vCJD and BSE to wild-type inbred mice. Lane 1, BSE-inoculated FVB mouse; lane 2, vCJD- inoculated FVB mouse; lane 3, BSE-inoculated C57BL/6 mouse; lane 4, BSE- inoculated SJL mouse; lane 5, vCJD-inoculated SJL mouse; lane 6, BSE-inoculated RπiS mouse. (H) Secondary transmission of vCJD and BSE in wild-type inbred mice. Lanes 1-4, BSE was passaged twice in C57BL/6 mice and then passaged in different wild-type mice: lane 1, C57BL/6 mouse; lane 2, FVB mouse; lane 3, SJL mouse; lane 4, RπiS mouse. Lanes 5 and 6, second passage of SJL-passaged BSE in further SJL mice (lane 5) or FVB mice (lane 6). Western blots were analysed by high-sensitivity ECL using biotinylated anti-PrP monoclonal antibody ICSM 35 (A-D, F-H) or 3F4 (E).

Figure 3

Scattergraph of proportions of protease-resistant PrP in higher molecular mass (diglycosylated) and low molecular mass (monoglycosylated) glycoforms seen in brain tissue from sporadic CJD, vCJD, BSE and in transgenic mice following challenge with CJD, vCJD and BSE. Data points are plotted as mean 6 SEM. Human cases, indicated as circles: sporadic CJD type 1 PrPSc, light grey (n = 12); sporadic CJD type 2 PrPSc, mid-grey (n = 49); sporadic CJD type 3 PrPSc, dark grey (n = 22); vCJD type 4 PrPSc, yellow (n = 16). Cattle BSE, black square (n = 3). Transmissions to 129MM Tg35 mice, upward triangles: sporadic CJD type 1 PrPSc-inoculated mice, blue (n = 7); vCJD type 4 PrPSc-inoculated mice, green (n = 10); BSEinoculated mice, red (n = 9; n = 1). Transmissions to 129MM Tg45 mice, inverted triangles: sporadic CJD type 2 PrPSc-inoculated mice, blue (n = 3); vCJD type 4 PrPSc- inoculated mice, green (n = 4); BSEinoculated mice, red (n = 4).

Figure 4 Scattergraph of proportions of protease-resistant PrP in higher molecular mass (diglycosylated) and low molecular mass (monoglycosylated) glycoforms seen in sporadic CJD, vCJD, BSE and in wild-type mice following challenge with vCJD and BSE. Data points are plotted as mean 6 SEM. (A-C) Human cases indicated as circles: sporadic CJD type 1 PrPSc, light grey (n = 12); sporadic CJD type 2 PrPSc, mid-grey (n = 49); sporadic CJD type 3 PrPSc, dark grey (n = 22); vCJD type-4 PrPSc, yellow (n = 16). Cattle BSE, black square (n = 3). (A) Primary transmission of vCJD and BSE to wild-type mice: vCJD-inoculated FVB mice, green diamond (n = 19); vCJD- inoculated SJL mice, green triangle (n = 4); BSE-inoculated FVB mice, red diamond (n = 12); BSE-inoculated SJL mice, red triangle (n = 7); BSE-inoculated RIIIS mice, red star (n = 4); BSE-inoculated C57BL/6 mice, inverted red triangle (n = 3). (B) Transmission of SJL-passaged BSE to further wild-type mice: SJL-passaged-BSE- inoculated FVB mice, blue diamond (n = 4); BSE passaged twice in SJL mice, blue triangle (n = 3); BSE passaged three times in SJL mice, open triangle (n = 3). (C) Transmission of BSE passaged twice in C57BL/6 mice to further wild-type mice: C57BL/6-passaged BSE to FVB mice, orange diamond (n = 3); C57BL/6-passaged BSE to SJL mice, orange triangle (n = 4); C57BL/6-passaged BSE to RIIIS mice, orange star (n = 3); C57BL/6-passaged BSE to C57BL/6 mice, inverted orange triangle (n = 3).

DETAILED DESCRIPTION OF THE INVENTION

TRANSGENIC NON-HUMAN ANIMAL

A "transgenic non-human animal", is an animal whose genome has been functionally altered by genetic manipulation, typically using recombinant DNA technology.

In the context of the present invention, this includes animals expressing, as a result of transgene expression, a PrP gene homozygous for M129.

As used herein, the term "transgenic non-human animal", is synonymous with the term "transgenic animal".

In transgenic animals, the term "gene" is synonymous with the term "transgene". The transgenic non-human animal of the present invention has incorporated into its genome a transgene. A transgene that has been incorporated into the genome of the transgenic non-human animal is a recombinant nucleic acid molecule that has stably integrated into the DNA of all of the germ cells and somatic cells. A transgene typically includes regulatory sequences, such as expression control sequences (e.g., promoters), which control the expression of the transgene in the cells of the animal.

Preferably, the transgenic non-human animal is a mammal. More preferably, the transgenic non-human animal is a rodent. More preferably, the transgenic non-human animal is a rat, hamster, rabbit, guinea pig or mouse. More preferably, the transgenic non-human animal is a mouse. Most preferably, the mouse is FVB/N x Svl29 x C57BL/6.

Preferably, the PrP gene - such as the murine PrP gene - is ablated (Bueler et al., 1992).

Any technique may be used to generate transgenic animals according to the invention. Preferably, the technique involves transfer of a transgene encoding methionine at codon 129 into single cell eggs of a strain of mice, for example, FVB/ N x Svl29 x C57BL/6, in which the murine PrP gene has been ablated (Bueler et al., 1992).

Methods for making transgenic animals are well known in the art and are described in Watson, J. D., et al., "The Introduction of Foreign Genes Into Mice," in Recombinant DNA, 2d Ed., W. H. Freeman & Co., New York (1992), pp. 255-272; Gordon, J. W., Intl. Rev. Cytol. 115:171-229 (1989); Jaenisch, R, Science 240: 1468-1474 (1989); and Rossant, J., Neuron 2: 323-334 (1990).

The human PrP gene according to the present invention may be introduced into the genome of mammals using any method for generating transgenic non-human animals known in the art. Embryonic target cells at various developmental stages may be used to introduce the transgene of the present invention. Different methods may be used depending on the stage of development of the embryonal target cell(s). These include, without limitation: 1. Microinjection of zygotes; Brinster, et al., Proc. Natl. Acad. Sci. (USA) 82: 4438-4442 (1985); 2. Viral integration; Jaenich, R, Proc. Natl. Sci. (USA) 73: 1260-1264; Jahner, et al, Proc. Natl. Acad. Sci. (USA) 82: 6927-6931 (1985); Van der Putten, et al, Proc. Natl. Acad. Sci. (USA) 82: 6148-6152 (1985); 3. Embryonal stem (ES) cells obtained from pre-implantation embryos that are cultured in vitro. Evans, M J., et al, Nature 292: 154156 (1981), Bradley, M. O., et al, Nature 309: 255-258 (1984); Gossler, et al., Proc. Natl. Acad. Sci. (USA) 83:9065-9069 (1986); Robertson et al., Nature 322: 445448 (1986).

The transgenic animals according to the invention may be employed for a variety of purposes.

The characteristics of prion infection in the transgenic animals of the present invention have parallels to prion infection in humans and thus have particular beneficial utility as a novel animal model of human prion infection, for example, vCJD.

In particular, mammalian models, for example, mice, may be of value in identifying and/or testing the ability of agents to modulate prion infection, as described herein

The animals into which a transgene is introduced may be an animal strain with defined mutations, or with a defined or undefined genetic background.

Typically, the transgenic animals will have a mixed genetic background with contributions from, for example, FVB/N, C57BL/6 and 129Sv inbred lines. Each transgenic animal may therefore have a different genetic background. Owing to this different genetic background, different responses may occur between transgenic animals, which are, like all transgenic lines, populations derived from single founders. For example, transgenic animals with one genetic background when challenged with BSE may show a type 2 or a type 4 response. Transgenic animals with a different genetic background when challenged with BSE may show a type 4 response. A person skilled in the art will be able to select transgenic non-human animals with a particular genetic background that give the desired response when contacted with a sample containing prions. The transgenic animals according to the present invention, may also be intercrossed with other animal strains with defined mutations, or with defined or undefined genetic backgrounds associated with prion infection. Comparison of the resulting progeny with or without the PrP transgene homozygous for Ml 29 may provide additional information on the alterations in occurrence, development, course, severity, progression, exacerbation, amelioration or cure of prion infection when expressed in these other genetic backgrounds. The transgenic animals according to the present invention, that are intercrossed may even be improved animal models.

PRION

As used herein the term "prion" refers to a proteinaceous infectious particle that lacks nucleic acid.

General teachings on prions are widely available in the art and are reviewed in, for example, Acta Neurobiol Exp (2002) 62, 153-66, FEBS Lett. (2002) 529, 17-21, Neurochem Int. (2002) 41, 353-5 and Med Clin North Am. (2002) 86, 551-71.

Preferably, the prions that are contacted with the transgenic animals of the present invention causes prion infection in mammals. More preferably, the prions that are contacted with the transgenic animals of the present invention cause prion infection in livestock ie. any farmed animal, for example, pigs, sheeps, cows or bulls. More preferably, the prions that are contacted with the transgenic animals of the present invention cause prion infection in cows. Most preferably, the prions that are contacted with the transgenic animals of the present invention cause prion infection in humans.

Preferably, the prions that are contacted with the transgenic animals of the present invention cause sCJD, vCJD or BSE in their natural hosts. More preferably, the prions that are contacted with the transgenic animals of the present invention cause vCJD or BSE in their natural hosts. Most preferably, the prions that are contacted with the transgenic animals of the present invention cause vCJD in their natural hosts. PrP GENE

The sequence of the PrP gene is avaiable in databases. For example, the nucleotide sequences of exon 1 and exon 2 of human PrP have the accession numbers X83415 and X83416. The protein sequence of human PrP has the accession number CAA58442.

A common polymorphism at codon 129 of the human PrP gene (PRNP), is a key determinant of susceptibility to sporadic and acquired prion diseases, and may affect age at onset in inherited prion disease (Baker et al., 1991; Collinge et al., 1991; Palmer et al, 1991). To date, all patients recognised with vCJD have been of the PRNP 129MM genotype (Collinge et al, 1996a; Zeidler et al, 1997).

PrP polymorphisms are known to affect prion strain propagation in mice and sheep Bruce, 1993). Similarly, codon 129 genotype may play a role in human prion strain propagation, since certain PrPSc types are closely associated with codon 129 genotypes. To date, types 1 and 4 PrPSc have been found only in individuals with the PRNP 129MM genotype and type 3 PrPSc only in genotypes MV or VV, while type 2 PrPSc is seen in association with all three genotypes (Collinge et al., 1996b; Wadsworth et al., 1999).

Preferably, the PrP gene homozygous for M129 is a human PrP gene.

The prions that are contacted with the transgenic animals of the present invention may have a PRNP genotype selected from PRNP129YN, PRNP129MV or PENP129MM.

Preferably, the prions that are contacted with the transgenic animals of the present invention have a PENP129MM genotype.

CHARACTERISTIC OF HUMAN PRION INFECTION

As used herein, the term "characteristic of human prion infection" means that the transgenic non-human animals according to the present invention develop a phenotype - such as a pathological (eg. neuropathological) and/or biochemical and/or molecular phenotype - that is associated with, attributed to, or characteristic of, human prion infection.

The characteristic of human prion infection may be characteristic of clinical prion infection.

Criteria for the clinical diagnosis of prion infection in mice are described by Carlson et al, 1986.

The characteristic of human prion infection may be characteristic of human sub- clinical prion infection.

Preferably, sub-clinical prion infection is characterised by the presence of human or bovine prion protein, for example, PrPSc, in the organs of transgenic non-human animals that accumulate prions during prion infection.

Therefore, the phenotype of the transgenic non-human animals may be associated with, attributed to, or characteristic of prion infection, for example, sCJD, vCJD or BSE.

Preferably, sub-clinical prion infection is characterised by the presence of human prion protein, for example, PrPSc in the organs of transgenic non-human animals that accumulate prions during prion infection. More preferably, the sub-clinical prion infection is characterised by the presence of human PrPSc in the brains (eg. the cerebral cortex and/or hippocampal regions) of transgenic non-human animals.

Therefore, the phenotype of the transgenic non-human animals may be associated with, attributed to, or characteristic of type 2 or type 4 PrPSc. Four human PrPSc types in brain tissue from patients with CJD have been described: types 1-3 are seen in classical (sporadic or iatrogenic) CJD, while type 4 is seen in vCJD (Collinge et al., 1996b). More preferably, the phenotype of the transgenic non-human animals is associated with, attributed to, or characteristic of type 4 PrPSc (ie. vCJD). More preferably, the phenotype of the transgenic non-human animals that is associated with, attributed to, or characteristic of vCJD is characterised by abundant PrP plaques which is an uncommon feature of prion disease in mice. Most preferably, the phenotype of the transgenic non-human animals that is associated with, attributed to, or characteristic of vCJD is characterised by abundant PrP plaques of the 'florid' type (a central plaque core surrounded by a ring of spongiform vacuoles), which are characteristic of vCJD in humans (Will et al. 1996), but rarely seen in mice.

Florid plaques were first described in Icelandic scrapie and have also been described in mice infected with the 111A scrapie strain (McBride et al., 1988). More recently, florid plaques have been reported in BSE-inoculated primates (Lasmezas et al, 1996) and in transgenic mice expressing ovine PrP infected with sheep-passaged BSE prions (Crozet et al., 2001).

Sub-clinical prion infection may be determined using various pathological and/or biochemical and/or molecular approaches that are apparent to a person skilled in the art, for example, histology, immunohistochemistry and/or Western blotting.

Preferably, immunohistochemistry is performed as follows. Mice are killed using CO₂ asphyxiation, brains fixed in 10 % buffered formol-saline and then immersed in 98% formic acid for 1 h and paraffin wax embedded. Serial sections of 4 μm are pre- treated with autoclaving, formic acid and 4 M guanidine thiocyanate. Abnormal PrP accumulation is examined using an anti-PrP monoclonal IgG antibody raised against recombinant human PrP, followed by a biotinylated anti-mouse IgG secondary antibody and an avidin-biotin-horseradish peroxidase conjugate before development with 3-,3-diaminobenzedine tetrachloride as the chromogen. The extent of gliosis is determined by GFAP (Dako) staining. Slides are pre-treated by heating in the microwave (900 W) in citrate buffer pH 6.0 for 25 min, followed by overnight incubation (1:1000). Biotinylated swine anti-rabbit immunoglobulins and avidin- biotin complex are applied as described above. Harris haematoxylin is used as the counterstain. Appropriate controls are used throughout.

Preferably, Western blotting is performed as follows. Preparation of brain homogenates (10% w/v in PBS), proteinase K digestion (50 or 100 mg of proteinase K for 1 h at 37°C) and subsequent Western blotting is performed as described previously (Wadsworth et al., 2001). For primary screening of both transgenic and wild-type mouse brain homogenates, blots are probed with a biotinylated anti-PrP monoclonal antibody which recognises both human and mouse PrP (biotinylated- ICSM 35) in conjunction with an avidin-biotin-alkaline phosphatase conjugate (Dako) and development in chemiluminescent substrate (CDP-Star; Tropix Inc.).

Glycoform ratio analysis may even be used which refers to the analysis of the different types of glycosylation of PrP^Sc i.e. the di-, mono- and un-glycosylated forms, as described by Baron et al. (1999) J. Clin. Microbiol. 37, 3701-3704. The molecular weight and relative intensity of each of the three glycoforms may be measured to calculate a glycoform ratio.

ASSAY METHOD

Mammalian models, for example, mice, may be of value in testing the ability of agents - such as pharmaceutical preparations of novel agents - to modulate prion infection. Transgenic animals according to the present invention are particularly useful in testing agents in this regard.

In addition to screening for unknown agents/compounds, the transgenic animals may be particularly useful in studies employing administration of natural or recombinant proteins, peptides or other agents or their derivatives already known or suspected to be involved in modulating prion infection or other natural or pharmacological agents already known to be active and/or of therapeutic value in these conditions. Agents that might be useful in treating prion infection are described in, for example, Nature (2001) 412, 739-43 and PNAS (2001) 98, 9836-41.

Agents/compounds identified as effective in such screening or analysis based on the use of transgenic animals according to the present invention may be particularly useful in the modulation of human prion infection and/or disorders related to prion infection with a view to delaying or preventing the occurrence, development, course, severity or progression of the infection, avoiding its exacerbation, and preferably promoting its amelioration or cure in mammals, or more preferably in humans. Compounds may also be developed based on screening or analysis in the transgenic animals of the present invention, which promote the occurrence, development, or progression of prion infection.

As used herein, the term "modulating" may refer to preventing, suppressing, alleviating, restorating or elevating or otherwise affecting a human prion infection.

Preferably, the term "modulating" refers to preventing, suppressing or alleviating human prion infection.

A transgenic non-human animal may be contacted with a sample containing prions. The term "sample" as used herein, has its natural meaning. A sample may be any physical entity containing one or more prions. The sample may be or may be derived from biological material. The sample may even comprise purified, or substantially purified prion protein.

Preferably, the sample is derived from the brain of an infected mammal, for example, a cow or a human, and may be or may be derived from a brain homogenate, for example, a brain homogenate of one or more brainstems.

The sample may be prepared by mixing with a solution. Preferably, the solution is a buffer such as phosphate buffered saline. The sample may be contacted with one or more transgenic animals according to the present invention that have been anaethetised using an anaesthetic such as halofhane/02-

Preferably, the method of contact of the one or more samples is via introduction of at least part of the sample into the brain of the transgenic animals, such as by injection. More preferably, the sample is injected into the right parietal lobe of the brain of the transgenic animals.

One or more agents may be contacted with the brain of the transgenic animals, such as by injection - such as into the right parietal lobe of the brain of the transgenic animals.

Typically, one or more agents are contacted with the transgenic animals by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, or subcutaneous injection or infusion, or implant), nasal, pulmonary, rectal, sublingual, or topical routes of administration, and can be formulated in dosage forms appropriate for each route of administration, e.g. in soluble form, suspension, or other suitable pharmaceutical formulations.

Optionally, the sample(s) and/or the agent(s) may be contacted with non-transgenic animals, for example, as controls. Preferably, one or more transgenic animals are contacted with the sample but are not been contacted with an agent.

The transgenic animals may be incubated following contact with the sample and/or contact with an agent. As used herein, the term "incubated" means the maintenance of the test animal in appropriate conditions, such as a containment facility as is well known in the art.

The effect of the agent on prion infection may be determined using various methods.

For example, transgenic animals may be monitored for symptoms of human clinical prion infection. At the onset of symptoms, the test animals may be examined regularly and may be culled if showing signs of distress. Criteria for clinical diagnosis of prion infection in mice, including examples of symptoms are described by Carlson (1986), Cell, 46, 503-511 and may include, among others, generalised tremor, ataxia or rigidity of the tail, or combinations thereof.

Preferably, transgenic animals have a prion infection characteristic of human sub- clinical prion infection. These transgenic animals may die apparently of age-related causes without clinical signs of prion disease at ages typical for inoculated or mock- inoculated mice.

Biopsies of the test animals may be performed to determine the effect of one or more agents on prion infection. The biopsy may be performed on any suitable organ or tissue such as one in which prions accumulate. Preferably, a brain biopsy is performed. Various methods well known in the art may be used for the detection of prion proteins - such as histology, immunohistochemistry and/or Western blotting, as described herein. Other methods, for example, electronic-property probing may even be used as described in WO 9831839.

Advantageously, the assay method of the present invention is used to identify agents that are capable of modulating prion infection.

Agents may restore, elevate, or increase human prion infection. Such agents may be identified in one or more transgenic animals in which the onset of symptoms is shorter in comparison to one or more control transgenic animals that have been contacted with the same sample but have not been contacted with an agent. Preferably, agents are identified in transgenic animals in which the amount of PrPSc - such as type 2 or type 4 PrPSc - is increased in comparison to one or more control transgenic animals that have been contacted with the same sample but have not been contacted with an agent as determined by, for example, histology, immunohistochemistry and/or Western blotting, as described herein.

Preferably, agents prevent, suppress, alleviate, or reduce prion infection. Such agents may be identified in one or more transgenic animals in which the onset of symptoms is longer (or even substantially prevented) in comparison to one or more control transgenic animals that have been contacted with the same sample but have not been contacted with an agent. Preferably, agents are identified in transgenic animals in which the amount of PrPSc - such as type 2 or type 4 PrPSc - is lower (or even substantially absent) in comparison to one or more control transgenic animals that have been contacted with the same sample but have not been contacted with an agent as determined by, for example, histology, immunohistochemistry and/or Western blotting, as described herein.

REGULATORY SEQUENCES

In some applications of the present invention, a polynucleotide is operably linked to a regulatory sequence which is capable of directing the expression of a coding sequence, for example, the PrP coding sequence, such as in vivo in the test animal. By way of example, the present invention may involve the use of regulatory sequences operably linked to one or more transgenes to modulate their expression.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.

The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site.

Enhanced expression of the polynucleotide encoding a polypeptide may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host and/or to provide for the inducible control of the expression of a polypeptide.

Promoter and enhancer sequences may be present in the transgene, which may increase the levels of expression in the transgenic animals.

The nucleotide sequence(s) may be operably linked to at least a promoter.

Aside from the promoter native to the gene encoding a polypeptide, other promoters may be used to direct expression of such a polypeptide. The promoter may be selected for its efficiency in directing the expression of such a polypeptide in the desired expression host.

In another embodiment, a constitutive promoter may be selected to direct the expression of a particular polypeptide. Such an expression construct may provide additional advantages since it circumvents the need to culture the expression hosts on a medium containing an inducing substrate.

Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are those which are obtainable from the fungal genes for xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi), alcohol dehydrogenase (AdhA), -amylase (amy), amyloglucosidase (AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3 -phosphate dehydrogenase (gpd) promoters.

Examples of strong yeast promoters are those obtainable from the genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase and triosephosphate isomerase.

Examples of strong bacterial promoters are the α-amylase and SP02 promoters as well as promoters from extracellular protease genes.

Hybrid promoters may also be used to improve inducible regulation of the expression construct.

The promoter may additionally include features to ensure or to increase expression in a suitable host. For example, the features may be conserved regions such as a Pribnow Box or a TATA box. The promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence. For example, suitable other sequences include the Shl-intron or an ADH intron. Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' signal sequence (see Sleat (1987), Gene 217, 217-225; and Dawson (1993), Plant Mol. BioL 23, 97).

NUCLEOTIDE SEQUENCE

Aspects of the present invention involve the use of nucleotide sequences, which may be available in databases. The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin. The nucleotide sequence may be double-stranded or single- stranded whether representing the sense or antisense strand or combinations thereof.

The nucleotide sequence may be DNA.

The nucleotide sequence may be prepared by use of recombinant DNA techniques (e.g. recombinant DNA).

The nucleotide sequence may be cDNA.

The nucleotide sequence may be the same as the naturally occurring form, or may be derived therefrom.

VARIANTS/HOMOLOGUES/DER]NATINES

The present invention also encompasses the use of variants, homologues and derivatives of any thereof.

Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".

In the present context, a homologous sequence is taken to include an amino acid sequence, which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence.

The amino acid sequences may also have deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include asp attic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

By way of example, conservative substitution of M include C, S and T.

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid , 7-amino heptanoic acid*, L-methionine sulfone*^*, L-norleucine*, L-norvaline*, p- nitro-L-phenylalanine*, L-hydroxyproline , L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino) , L- Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (l,2,3,4-tetrahydroisoquinoline-3- carboxyl acid)*, L-diaminopropionic acid ^# and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β- alanine residues. A further form of variation involves the presence of one or more amino acid residues in peptoid form will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al, PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

Nucleotide sequences may include within them synthetic or modified nucleotides.

CONSTRUCTS

The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - may include a nucleotide sequence directly or indirectly attached to a promoter. The term "fused" includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment. VECTORS

The term "vector" includes expression vectors and transformation vectors and shuttle vectors.

The term "expression vector" means a construct capable of in vivo or in vitro expression.

The term "transformation vector" means a construct capable of being transferred from one entity to another entity - which may be of the species or may be of a different species. If the construct is capable of being transferred from one species to another - such as from an Escherichia coli plasmid to a bacterium, such as of the genus Bacillus, then the transformation vector is sometimes called a "shuttle vector". It may even be a construct capable of being transferred from an E. coli plasmid to an Agrobacterium to a plant.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.

Vectors may contain one or more selectable marker genes.

Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

Polynucleotides may be incorporated into a recombinant vector (typically a replicable vector), for example a cloning or expression vector.

TREATMENT

It is to be appreciated that all references herein to treatment refer to the modulation of prion infection.

The treatment may be of mammals such as livestock and/or humans. ANTIBODY

An agent for use in the composition may comprise one or more antibodies.

The "antibody" as used herein includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in US-A- 239400. Neutralising antibodies, i.e., those, which inhibit biological activity are especially preferred for diagnostics and therapeutics.

Antibodies may be produced by standard techniques, such as by immunisation with the substance of the invention or by using a phage display library.

THERAPY

Agents identified by the method of the present invention may be used as therapeutic agents - i.e. in therapy applications.

As with the term "treatment", the term "therapy" includes curative effects, alleviation effects, and prophylactic effects.

The therapy may be on mammals such as humans or livestock.

The therapy may be for treating conditions caused by or associated with prion infection.

AGENT As used herein, the term "agent" may be a single entity or it may be a combination of entities.

The agent may be an organic compound or other chemical. The agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial. The agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof. The agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule. The agent may even be an antibody.

The agent may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.

By way of example, the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised agent, a peptide cleaved from a whole protein, or a peptides synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof, a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.

Typically, the agent will be an organic compound. Typically the organic compounds will comprise two or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. For some applications, preferably the agent comprises at least one cyclic group. The cyclic group may be a polycyclic group, such as a non-fused polycyclic group. For some applications, the agent comprises at least the one of said cyclic groups linked to another hydrocarbyl group.

The agent may contain halo groups. Here, "halo" means fluoro, chloro, bromo or iodo.

The agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.

The agent may be in the form of a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof. For a review on suitable salts see Berge et al, J. Pharm. Sci., 1977, 66, 1-19.

The agent of the present invention may be capable of displaying other therapeutic properties.

The agent may be used in combination with one or more other pharmaceutically active agents.

If combinations of active agents are administered, then they may be administered simultaneously, separately or sequentially.

STEREO AND GEOMETRIC ISOMERS

The agents may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of the entire individual stereoisomers and geometric isomers of those agents, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree). PHARMACEUTICAL SALT

The agent may be administered in the form of a pharmaceutically acceptable salt.

Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example include those mentioned by Berge et al, in J. Pharm. Sci., 66, 1-19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p-toluenesulphonate salts.

When one or more acidic moieties are present, suitable pharmaceutically acceptable base addition salts can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and pharmaceutically-active amines such as diethanolamine, salts.

A pharmaceutically acceptable salt of an agent may be readily prepared by mixing together solutions of an agent and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.

An agent may exist in polymorphic form.

An agent may contain one or more asymmetric carbon atoms and therefore exist in two or more stereoisomeric forms. Where an agent contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur. The present invention includes the individual stereoisomers of an agent and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.

Separation of diastereoisomers or cis- and tra/zs-isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of an agent or a suitable salt or derivative thereof. An individual enantiomer of an agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.

The present invention also encompasses all suitable isotopic variations of an agent or a pharmaceutically acceptable salt thereof. An isotopic variation of an agent or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that may be incorporated into an agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶C1, respectively. Certain isotopic variations of an agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as ³H or ¹⁴C is incorporated are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be prefened in some circumstances. Isotopic variations of an agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

It will be appreciated by those skilled in the art that an agent may be derived from a prodrug. Examples of prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form an agent of the present invention which are pharmacologically active. It will be further appreciated that certain moieties known as "pro-moieties", for example as described in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985 (the disclosured of which is hereby incorporated by reference), may be placed on appropriate functionalities of agents. Such prodrugs are also included within the scope of the invention.

The present invention also includes the use of zwitterionic forms of an agent of the present invention.

SOLVATES

The present invention also includes the use of solvate forms of an agent of the present invention.

PRO-DRUG

As indicated, the present invention may also include the use of pro-drug forms of an agent.

PHARMACEUTICALLY ACTIVE SALT

An agent may be administered as a pharmaceutically acceptable salt. Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.

CHEMICAL SYNTHESIS METHODS

An agent may be prepared by chemical synthesis techniques.

It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional techniques, for example as described in "Protective Groups in Organic Synthesis" by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by P.J.Kocienski, in "Protecting Groups", Georg Thieme Verlag (1994).

It is possible during some of the reactions that any stereocentres present could, under certain conditions, be racemised, for example if a base is used in a reaction with a substrate having an optical centre comprising a base-sensitive group. This is possible during e.g. a guanylation step. It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection/deprotection regimes, etc. as is well-known in the art.

The compounds and salts of the invention may be separated and purified by conventional methods.

Separation of diastereomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compound of formula (I) or a suitable salt or derivative thereof. An individual enantiomer of a compound of formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the conesponding racemate using a suitable chiral support or by fractional crystallisation of the diastereomeric salts formed by reaction of the conesponding racemate with a suitably optically active acid or base.

An agent or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesize an agent in whole or in part. For example, if they are peptides, then peptides may be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).

Synthesis of peptide agents may be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising an agent or any part thereof may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant agent.

In an alternative embodiment of the invention, the coding sequence of a peptide agent (or variants, homologues, derivatives, fragments or mimetics thereof) may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).

MIMETIC

As used herein, the term "mimetic" relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a reference agent.

CHEMICAL DERΓVATΓVE

The term "derivative" or "derivatised" as used herein includes chemical modification of an agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.

CHEMICAL MODIFICATION

The chemical modification of an agent may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the agent and the target.

In one aspect, the identified agent may act as a model (for example, a template) for the development of other compounds.

PHARMACEUTICAL COMPOSITIONS Pharmaceutical compositions useful in the present invention may comprise a therapeutically effective amount of agent(s) and pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

Pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent may be selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).

Preservatives, stabilizers, dyes and even flavoring agents may be provided in pharmaceutical compositions. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

There may be different composition formulation requirements dependent on the different delivery systems. By way of example, pharmaceutical compositions useful in the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be administered by a number of routes.

Agents may also be used in combination with a cyclodextrin. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes. As an alternative to direct complexation with the drug the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser. Alpha-, beta- and gamma- cyclodextrins are most commonly used and suitable examples are described in WO-A- 91/11172, WO-A-94/02518 and WO-A-98/55148.

If an agent is a protein, then said protein. may be prepared in situ in the subject being treated. In this respect, nucleotide sequences encoding said protein may be delivered by use of non- viral techniques (e.g. by use of liposomes) and/or viral techniques (e.g. by use of refroviral vectors) such that the said protein is expressed from said nucleotide sequence.

ADMINISTRATION

The term "administered" includes delivery by viral or non-viral techniques. Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, refroviral vectors, lentiviral vectors, and baculoviral vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.

The components useful in the present invention may be administered alone but will generally be administered as a pharmaceutical composition - e.g. when the components are in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the components may be administered (e.g. orally) in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

If the pharmaceutical is a tablet, then the tablet may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpynolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Prefened excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The routes for administration (delivery) include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual.

It is to be understood that not all of the components of the pharmaceutical need be administered by the same route. Likewise, if the composition comprises more than one active component, then those components may be administered by different routes.

If a component is administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the component; and/or by using infusion techniques.

For parenteral administration, the component is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

As indicated, the component(s) useful in the present invention may be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A™) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA™), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent and a suitable powder base such as lactose or starch.

Alternatively, the component(s) may be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The component(s) may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route. For ophthalmic use, the compounds may be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the component(s) may be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, it may be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

DOSE LEVELS

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.

FORMULATION

The component(s) may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.

IDENTIFICATION OF GENES

hi another aspect, the present invention relates to methods for the identification of genes associated with prion infection, including susceptibility to prion infection.

By way of example, the genes of transgenic animals according to the present invention and animals not comprising the PrP gene homozygous for Ml 29 are compared by various methods known to persons skilled in the art, such as subtractive hybridisation (Sambrook et al. (1989) Konietzko U & Kuhl D (1998). Nucleic Acids Res 26, 1359-61) and genome scanning (Stephenson et al. (1960) Genomics 69, 47- 53). By using these methods, regions of DNA that are different are identified. The genes involved in prion infection may then be identified. In subtractive hybridisation, cDNA is processed so that the cDNA becomes highly enriched for sequences present only in the trangenic animals. The enriched cDNA may then be used to screen a cDNA library for clones that have sequences homologous to those of the enriched cDNA. The screening may be performed using a number of methods well known in the art such as nucleic acid hybridisation or with PCR probes.

The specificity of the probe i.e. whether it is derived from a highly conserved, conserved or non-conserved region and the stringency of the hybridisation or amplification (high, intermediate or low) will determine whether the probe identifies only naturally occurring coding sequences, or related sequences.

In genomic screening, genomic DNA from repeating DNA segments (called microsatellite DNA) is amplified by PCR from the trangenic animals and animals not comprising the PrP gene homozygous for M129. Linkage analysis is then performed to determine the relative positions of the genes on the chromosomes.

Preferably, the PCR primers used for amplifying microsatellite DNA are selected from commercially available kits such as the M13-tailed mouse genome-wide screening set (Research Genetics, Huntsville, AL). PCR reactions are performed in

96 well plates using for example, radioactively labeled primers. The PCR products are then resolved by denaturing gel electrophoresis and the products detected using for example, autoradiography. Marker genes that have known locations on chromosomes are compared in the trangenic animals and animals not comprising the

PrP gene homozygous for Ml 29 and those that show suggestive or significant linkage are subjected to linkage analysis. This can be employed using a suitable software package such as Map Manager QT (Manly & Olson et al. 1999, Mamm. Genome 10

327-334). The location of any genes that differ between the trangenic animals and ammals not comprising the PrP gene homozygous for M129 may then be mapped.

PCR as described in US 4683195, US 4800195 and US 4965188 provides additional uses for oligonucleotides based upon target sequences. Such oligomers are generally chemically synthesized, but they may be generated enzymatically or produced from a recombinant source. Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'->3') and one with antisense (3'<-5') employed under optimised conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantification of closely related DNA or RNA sequences.

Probes may also be used for mapping the endogenous genomic sequence related to prion infection. The sequences may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in situ hybridisation to chromosomal spreads (Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City), flow-sorted chromosomal preparations, or artificial chromosome constructions such as YACs, bacterial artificial chromosomes (BACs), bacterial PI constructions or single chromosome cDNA libraries.

In situ hybridisation of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers are invaluable in extending genetic maps. Examples of genetic maps can be found in Science (1995; 270:410f and 1994; 265:1981f). Often the placement of a gene on the chromosome of another species may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms or parts thereof, by physical mapping. This provides valuable information when searching for genes associated with orion infection using positional cloning or other gene discovery techniques. Once the genes of interest have been crudely localised by genetic linkage to a particular genomic region any sequences mapping to that area may represent associated or regulatory genes for further investigation.

Optionally, the function of the genes associated with prion infection and/or susceptibility could be determined by first sequencing the DNA of the said gene(s) using methods well known in the art. The sequence(s) could then be compared with DNA sequences available on databases to identify homologous sequences. Using this information, the function of the homologous sequences could be determined. The consistent distinctive strain propagation in particular strains of transgenic mice — such as FVB and C57BL/6 versus SJL and RIIIS lines - may even allow mapping of genes relevant to strain selection and propagation.

DIAGNOSIS

In still another aspect, the present invention relates to methods for the diagnosis of prion infection. Advantageously, the use of transgenic non-human animals according to the present invention, means that prions that cause various prion diseases - such as BSE, vCJD or sCJD may be identified using the same strain of mice.

Such a method for the detection of prions in a sample may comprise the steps of: (a) contacting one or more transgenic non-human animals expressing, as a result of transgene expression, a PrP gene homozygous for M129, with the sample; (b) incubating the transgenic non-human animals; (c) incubating the transgenic non- human animals; and optionally (d) performing a biopsy on the transgenic non-human animals for evidence of prions.

In accordance with this aspect of the invention, the transgenic non-human animals may be monitored for symptoms of. prion infection by examination for the development of symptoms of prion infection. At the onset of symptoms, the transgenic non-human animals are examined regularly and may be culled if showing signs of distress. Criteria for clinical diagnosis of prion infection in mice, including examples of symptoms are described by Carlson (1986), Cell, 46, 503-511 and include, among others, generalised tremor, ataxia or rigidity of the tail, or combinations thereof.

However, the transgenic non-human animals may develop sub-clinical infection without any obvious symptoms. Therefore, biopsies of the transgenic non-human animals may also be performed. The biopsy may be performed on any suitable organ or tissue such as one in which prions accumulate. Preferably, a brain biopsy is performed. Various methods well known in the art may be used for the detection of prion proteins, as described herein, - such as Western blotting (Collinge et al. 1996, Nature 383, 685-690), immunoassay (described in WO 9837210) and electronic- property probing (described in WO 9831839).

A person skilled in the art will be aware that it is necessary to use a sufficient number of test animals per sample.

The transgenic non-human animals according to the present invention, may be intercrossed with animal strains that have a short prion incubation time ie. the amount of time that elapses from contacting the transgenic non-human animal with a sample, to the time when the transgenic non-human animal first develops prion infection. An example of an animal strain with a short prion incubation time is SJL as described in WO 02/059363.

GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES

The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in careying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES

Example 1

Materials and methods

Generation of transgenic mice

The 759 bp human PrP ORF was amplified by PCR with Pfu polymerase from genomic DNA encoding methionine at codon 129, using forward primer 5'- GTCGACCAGTCATTATGGCGAACCTT-3 ' and reverse primer 5'- CTCGAGAAGACCTTCCTC ATCCC ACT-3 ' . Restriction sites Sail and Xhol (underlined) were introduced in the forward and reverse primers, respectively, for cloning. The sequence was confirmed and ligated into the cosmid vector CosSHaTet (Scott et al., 1989). Microinjection of the purified DNA was carried out according to the standard protocol into single cell eggs of a strain of mice (FVB/ N x Svl29 x C57BL/6) in which the murine PrP gene has been ablated (Bueler et al., 1992). Genotyping was performed by PCR, and PrP expression levels estimated by western blot analysis.

Transmission studies

Strict biosafety protocols were followed. Inocula were prepared, using disposable equipment for each inoculum, in a microbiological containment level 3 laboratory and inoculations perfonned within a class 1 microbiological safety cabinet. Five separate BSE inocula, each derived from single natural BSE-affected cow brainstems (1060, 1062, 1064, 1066, 1783), and a separate inoculum prepared from a pool of five natural BSE brainstems (1038) were studied. Aliquots of these (except 1783) have been used in previously published studies (Collinge et al., 1995; Hill et al:, 1997). BSE tissues were collected under strict aseptic conditions using sterile instrumentation, specifically for transmission studies, by the UK Central Veterinary Laboratory [now the Veterinary Laboratories Agency (VLA)]. The BSE pool homogenate was titrated into RIII wild-type mice at VLA with a resultant titre of 10^3'3 mouse intracerebral LD50 units/g of tissue. Sporadic and vCJD inocula were prepared from brain tissue from neuropatho logically confirmed cases. Consent for use of tissues for research was obtained. The genotype of each transgenic mouse was confirmed by PCR of tail DNA prior to inclusion and all mice were uniquely identified by sub-cutaneous transponders. RIIIS/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and SJL/OlaHsd mice were obtained from Harlan UK Ltd (Bicester, UK). Disposable cages were used, and all cage lids and water bottles were also uniquely identified by transponder and remained with each cage of mice throughout the incubation period. Care of the mice was according to institutional guidelines. Both transgenic and wild-type mice were anaesthetized with a mixture of halo thane and 02, and intracerebrally inoculated into the right parietal lobe with 30 ml of a 1% brain homogenate prepared in PBS. Thereafter, all mice were examined daily for clinical signs of prion disease. Mice were killed if they were exhibiting any signs of distress or once a diagnosis of prion disease was established. Criteria for clinical diagnosis of scrapie in mice were as described previously (Carlson et al., 1986).

Neuropathology and immunohistochemistry

Mice were killed using C02 asphyxiation, brains fixed in 10% buffered formol-saline and then immersed in 98% formic acid for 1 h and paraffin wax embedded. Serial sections of 4 mmwere pre-treated with autoclaving, formic acid arid 4 M guanidine thiocyanate. Abnormal PrP accumulation was examined using an anti-PrP monoclonal IgG antibody raised against recombinant human PrP (ICSM 35), followed by a biotinylated anti-mouse IgG secondary antibody and an avidin-biotin-horseradish peroxidase conjugate before development with 3',3-diaminobenzedine tetrachloride as the chromogen. The extent of gliosis was determined by GFAP (Dako) staining. Slides were pre-treated by heating in the microwave (900 W) in citrate buffer pH 6.0 for 25 min, followed by overnight incubation (1:1000). Biotinylated swine anti-rabbit im unoglobulins and avidin-biotin complex were applied as described above. Harris haematoxylin was used as the counterstain. Appropriate controls were used throughout.

Wester?ι blotting

Preparation of brain homogenates (10% w/v in PBS), proteinase K digestion (50 or 100 mg of proteinase K for 1 h at 37°C) and subsequent western blotting were performed as described previously (Wadsworth et al, 2001). For primary screening of both transgenic and wild-type mouse brain homogenates, blots were probed with a biotinylated anti-PrP monoclonal antibody which recognizes both human and mouse PrP (biotinylated-ICSM 35) in conjunction with an avidin-biotin-alkaline phosphatase conjugate (Dako) and development in chemiluminescent substrate (CDP-Star; Tropix Inc.). Primary screening of brain homogenates was performed blind to sample identity.

Quantitation and analysis of PrP glycoforms

Western blotting was performed as above but using different primary and secondary detection reagents. For transgenic mice expressing human PrP, blots were incubated with anti-PrP monoclonal antibody 3F4 (Kascsak et al., 1987), whereas for wild-type mice expressing mouse PrP, blots were incubated with anti-PrP monoclonal antibody 6H4 (Prionics, Switzerland), followed by incubation with goat anti-mouse IgG- alkaline phosphatase conjugate (Sigma) and development in chemifluorescent substrate (AttoPhos; Promega) and visualization on a. Storm 840 Phosphorlmager (Molecular Dynamics). Quantitation of PrPSc glycoforms was performed using ImageQuaNT software (Molecular Dynamics).

Example 2

Susceptibility of transgenic mice expressing human PrP Ml 29 to human and bovine prions

Transgenic mice were produced homozygous for a human PrP Ml 29 transgene anay and murine PrP null (Bueler et al., 1992) alleles (PrnpO/0), designated Tg(HuPrP129M+/+ Prnp0/0)-35 (129MM Tg35), with expression levels of human PrP two times that of pooled normal human brain (data not shown). These mice were challenged with prions from cases of sporadic CJD, vCJD and BSE. 129MM Tg35 mice were highly susceptible to prions from patients with sporadic CJD of the PRNP 129MM genotype, but were less susceptible to classical CJD prions from individuals of the PRNP 129VV genotype (Table I). Transmission of sporadic CJD of the PRNP 129MV genotype was associated with either consistent short-duration characteristics as with MM cases (1024) or long and variable incubation "periods (1020). This may reflect stochastic propagation of either 129M or 129V PrPSc in these patients. This was in contrast to Tg(HuPrP129V+/+ Prnp0/0)-152 mice, expressing human PrP V129 (129VV Tgl52), which, as we have reported previously (using the same inocula), are highly susceptible to classical CJD prions from all three PRNP genotypes (Collinge et al, 1995; Hill et al., 1997). The presence of a transmission barrier can be estimated by measuring the fall in mean incubation period on primary and second passage in the same host. Second passage of prions from sporadic CJD (I1202)-inoculated 129MM Tg35 mice resulted in an incubation period of 249 6 3 days (4/4 mice), which was not lower than primary passage [229 6 5 days (8/8 mice)]. It is possible that the small increase in incubation period reflects a lower prion titre in mouse than human brain since affected mice are culled at an early clinical stage. Consistent short incubation periods on primary passage with 100% attack rate and no fall in incubation period on second passage of CJD in these mice, as with our earlier studies with Tgl52 mice (Collinge et al., 1995), are consistent with lack of a transmission barrier to classical CJD 129MM prions. However, as with 129VV Tgl52 mice (Hill et al., 1997), 129MM Tg35 mice were much more resistant to vCJD 129MM prions, with only 1/14 mice succumbing to clinical prion disease at a prolonged incubation period (690 days) (Tables I and II). Indeed, as judged by development of clinical disease, 129MM Tg35 mice, expressing human PrP 129M, appeared less susceptible to vCJD than 129VV Tgl52 mice, expressing human PrP 129V (Hill et al, 1997). Similarly, 129MM Tg35 mice appeared highly resistant to BSE prions, with 6/49 clinically scored transmissions at variable and prolonged incubation periods (338-492 days) (Tables I and II).

Example 3

Sub-clinical infection in mice expressing human PrP Ml 29

While by clinical criteria these data might be interpreted as consistent with the existence of a substantial species barrier between cattle BSE and transgenic mice expressing 129M human PrP, we investigated all these mice for evidence of sub- clinical infection. We and others have previously demonstrated extensive sub-clinical prion infection in mice inoculated with a strain of hamster prions (Sc237 or 263K) thought to be non-pathogenic to wild-type mice (Hill et al., 2000; Race et al., 2001), questioning current definitions of transmission barriers, which have been conventionally assessed on the basis of occunence of clinical disease in inoculated animals. Surprisingly, as assessed by histology, immunohistochemistry and/or the presence of human PrPSc on western blotting of brain tissue, we found that all (13/13) vCJD inoculated 129MM Tg35 mice, which had died apparently of age-related causes without clinical signs of prion disease at ages typical for uninoculated or mock inoculated mice, had pathological (Figure IA) and/or biochemical (Figure 2 A) evidence of prion infection. Only a single (1/14) (Table II) 129MM Tg35 mouse challenged with vCJD developed clinical prion disease. Excluding those animals that died soon after inoculation, which were not examined further, all other mice (13/14), which died of intercurrent or age-related illness between 342 and 726 days post- inoculation without showing clinical signs of prion disease, were positive for type 4 PrPSc in their brains. There appeared, therefore, to be a 100% infection rate of vCJD- inoculated 129MM Tg35 mice (Table I). A smaller proportion (8/49) of BSEinoculated 129MM Tg35 mice also developed sub-clinical disease (Table I; Figure 2D). Widespread sub-clinical disease was not seen, however, in vCJD- or BSE- inoculated 129VV Tgl52 mice (Hill et al, 1997). Since the methods used for PrPSc detection in the current study are more sensitive than those used in our earlier study (Hill et al., 1997), we reanalysed the 129VV Tgl52 mouse brains (16) for which frozen tissue remained for study, using the same methods used here, and found no PrPSc. Sub-passage of infectivity from both 129V and 129M human PrP-expressing lines of transgenic mice will be necessary to further characterise and quantitate these transmission barriers.

Example 4

Transgenic mice expressing human PrP Ml 29 develop the neuropathological features and PrPSc type of vCJD following inoculation with BSE or vCJD prions

Inoculation of vCJD prions into 129MM Tg35 mice resulted in clinical disease in only a single mouse, but widespread sub-clinical disease with human PrPSc readily detectable in brain by Western blot analysis. In previous transmission studies of vCJD prions to 129VV Tgl52 mice, a novel type 5 PrPSc pattern was obtained, and thought to represent a prion strain switch resulting from mismatch of the codon 129 polymorphism in inoculum and host human PrP (Hill et al., 1997). A prediction of the hypothesis that prion strain type is encoded by PrPSc structural properties, and that the PRNP codon 129 polymorphism plays a key role in human prion strain propagation, is that transmission of vCJD prions (containing human PrPSc type 4) to 129MM Tg35 mice would result in faithful propagation of type 4 PrPSc. This was indeed what we observed: the PrPSc type seen, as judged by PrPSc fragment sizes (Figure 2A, compare lanes 1 and 2), was the type 4 pattern characteristic of vCJD prions in human brain. The glycoform ratio also closely resembled that of type 4 PrPSc in human brain (Figure 3). As we have reported previously, a small difference is seen on glycoform ratios of the same prion strain propagated in mice and human brain, presumably reflecting the superimposition of species-specific glycosylation effects on the prion strain specific pattern (Hill et al., 1997). Furthermore, the neuropathological features in the vCJD-inoculated 129MM Tg35 mice were quite different from those of 129VV Tgl52 mice propagating type 5 human PrPSc, where no PrP immunoreactive plaques were seen (Hill et al., 1997). Remarkably, the vCJD- inoculated 129MM Tg35 mice not only developed abundant PrP plaques, an uncommon feature of prion disease in mice, but many of these were of the "florid' type (a central plaque core surrounded by a ring of spongiform vacuoles), which are characteristic of vCJD in humans (Will et al., 1996) (Figure IA) but rarely seen in mice. Florid plaques were first described in Icelandic scrapie and have also been described in mice infected with the 111A scrapie strain (McBride et al., 1988). More recently, florid plaques have been reported in BSE-inoculated primates (Lasmezas et al., 1996) and in transgenic mice expressing ovine PrP infected with sheep-passaged BSE prions (Crozet et al., 2001). BSE prion inoculation of 129MM Tg35 mice also resulted in both clinical disease and sub-clinical infection (Tables I and II). In sharp contrast to BSE transmission to 129VV Tgl52 mice, where we were unable to detect PrPSc in brain (Hill et al., 1997), PrPSc was readily detectable in brains of clinically sick 129MM Tg35 mice and in mice not showing clinical signs of prion disease when they died at advanced age (Figures 2C and 3). In one of the eight sub-clinically affected mice, type 4 human PrPSc was seen (Figure 2C, lane 2), indistinguishable from that seen in vCJD-inoculated 129MM Tg35 mice and in human vCJD itself. In this mouse, neuropathological features were identical to those of vCJD-inoculated mice, with abundant florid plaques as in human vCJD (Figure IB). These data further supported the conclusion that vCJD is caused by a BSE-like prion strain. However, in all other sub-clinically affected BSE-inoculated 129MM Tg35 mice (7/8), an alternate phenotype was observed (Table II). This was also seen in all clinically affected BSEinoculated 129MM Tg35 mice where brain was available for analysis (5/6) (Table H).

Example 5

Some Tg(HuPrPM129) mice develop a distinct phenotype following inoculation with BSE prions

In 4/6 clinically affected and 6/8 sub-clinically affected BSE-inoculated 129MM Tg35 mice, a distinctive human PrPSc type was seen with a quite different fragment size of unglycosylated PrPSc following proteinase K digestion and a different ratio of the three glycoforms, monoglycosylated PrPSc being most abundant (in marked contrast to type 4 PrPSc, where diglycosylated PrPSc predominates) (Figure 2B, C and E, compare lanes 1 and 2). Comparison with known human PrPSc types in CJD indicated that this type conesponded, both with respect to fragment sizes and glycoform ratio, to the type 2 PrPSc seen in sporadic and iatrogenic CJD (Figure 2D, compare lanes 1 and 2, and Figure 3). Human PrPSc types can also be distinguished by their metal-binding properties. Both type 1 and type 2 human PrPSc undergo a shift in fragment size following proteinase K treatment if treated with the metal chelator EDTA (Wadsworth et al, 1999). Type 3 (also seen in classical CJD) and type 4 PrPSc do not undergo a metaldependent shift in proteinase K cleavage site on treatment with EDTA (Wadsworth et al., 1999). Treatment of BSE inoculated 129MM Tg35 mouse brain homogenates with EDTA prior to proteinase K cleavage demonstrated that while that showing a type 4 pattern was unaltered by this treatment (data not shown), those showing a type 2 pattern underwent the expected band shift indistinguishable from type 2 PrPSc from CJD-affected human brain (Figure 2E, compare lanes 1 and 2 with 4 and 5). Routinely, in our transmission studies, individual mouse brains from a group are either frozen for biochemical studies or fixed for histology; in some mice, one hemisphere is frozen and the other fixed to allow both techniques on an individual mouse. Histopathological analysis on fixed tissue and biochemical analysis on frozen tissue was only available on a single animal showing type 2 PrPSc. However, neuropathological features of this subclinically affected, BSE-inoculated 129MM Tg35 mouse, showing a type 2 PrPSc pattern in the brain, were quite distinct from those with type 4 PrPSc. There was no specific PrP immunoreactivity; in particular, there were no florid or other plaques (Figure 1G). However, widespread neuronal vacuolation (Figure IE) and extensive gliosis (Figure IF), consistent with spongiform encephalopathy, clearly confirm sub-clinical disease in this mouse. While vCJD prions produce a neuropathological pattern in 129MM Tg35 mice similar to that seen in human vCJD, and the characteristic PrPSc type of vCJD is maintained in all mice, BSE inoculation results in two distinct but highly consistent phenotypes: one indistinguishable from the vCJD transmissions, and associated with the characteristic molecular 'signature' of BSE; and a second that resembles transmission of the commonest molecular sub-type of classical CJD.

Example 6

vCJD and BSE transmission to a further HuPrP 129M-expressing transgenic line

We also inoculated a second transgenic line expressing HuPrPM129, generated as described for Tg35, with vCJD and BSE prions. Tg(HuPrP129M+/+ Prnp0/0)-45 (129MM Tg45) mice were produced similarly to 129MM Tg35 mice, but have a level of expression of human PrP 4-fold higher than a pooled normal human brain standard (data not shown). These mice were also highly susceptible to sporadic CJD, with a 100% attack rate, extremely short and consistent incubation periods (1024: 7/7 mice developed disease with an incubation time of 155 ±5 days), and no fall in incubation period on second passage, consistent with lack of a transmission barrier to classical CJD prions. Again, as judged by clinical disease, we found that these animals were much less susceptible to vCJD and BSE. However, as seen with BSE- or vCJD inoculated 129MM Tg35 mice, evidence of sub-clinical prion infection was seen in most clinically unaffected mice (Table II). While only 1/4 vCJD-inoculated 129MM Tg45 mice developed clinical disease (at 580 days), the remaining 3/4 mice had neuropathological and biochemical evidence of prion infection. Again, in close agreement with the results from 129MM Tg35 mice, analysis of brains of vCJD- inoculated 129MM Tg45 mice consistently revealed widespread florid plaque deposition (Figure 1C) and type 4 PrPSc (Figure 2F, lane 2 and Figure 3). Similarly, none of the BSE-inoculated 129MM Tg45 mice developed clinical signs of prion disease for >700 days, but 9/12 had sub-clinical prion infection (Table II). Neuropathological examination of BSE inoculated 129MM Tg45 mice revealed closely similar pathological findings to that of vCJD-inoculated 129MM Tg45 mice with florid plaques (Figure ID) and western blot analysis of brain tissue revealed type 4 PrPSc (Figure 2F, lane 3 and Figure 3). To date, the alternate neuropathological pattern associated with type 2 PrPSc has not been detected in BSE-inoculated 129MM Tg45 mice.

Example 7

vCJD and BSE transmission to various inbred lines of non-transgenic mice

BSE prions transmit readily to wild-type mice but with prolonged and variable incubation periods. We have previously reported the PrPSc type of both FVB and C57BL/6 mice when inoculated with BSE (Collinge et al., 1996b; Hill et al, 1997). As with other species naturally or experimentally infected with BSE that we have reported, a BSE-like pattern is produced with a characteristic PrPSc fragment size and glycoform ratio. However, these transmissions involve PrP from another mammalian species of different molecular mass, such that the proteins are not directly comparable, as with transmissions of human prion disease to transgenic mice expressing only human PrP. This mouse PrPSc pattern is, therefore, refened to as "diglycosylated dominant'. In independent experiments to determine the range of incubation periods of BSE in many inbred mouse lines, as part of studies to map prion incubation time genes (Lloyd et al., 2001), we have identified two inbred lines of mice in which BSE transmission is associated with the production of a distinctive PrPSc type, with PrPSc glycoform ratios closely similar to that of human sporadic CJD and refened to here as a 'monoglycosylated dominant' PrPSc pattern. Interestingly, these lines are also associated with unusually short incubation periods for BSE (Table III). All four inbred mouse lines have the same Prnp coding sequence (Prnp-a; data not shown) and are homozygous for methionine at codon 128, the conesponding murine codon to PRNP codon 129. Following inoculation with BSE prions, both FVB and C57BL/6 mice show the characteristic diglycosylated dominant PrPSc pattern in the brain (Figures 2G, lanes 1 and 3, and 4A). However, when inoculated with the same BSE inoculum, SJL and RIIIS mice exhibit a monoglycosylated dominant PrPSc pattern (Figures 2G, lanes 4 and 6, and 4A). This PrPSc type is stable on further passage to both SJL and FVB mice (Figures 2H, lanes 5 and 6, and 4C) and is unaffected by EDTA treatment (data not shown). The propagation of the monoglycosylated PrPSc glycoform pattern is established by the host in which primary passage is carried out, as both SJL and RIIIS mice are capable of propagating the diglycosylated dominant PrPSc pattern when challenged with BSE passaged twice in a C57BL/6 mouse (Figures 2H, lanes 3 and 4, and 4C). vCJD prions behave in the same way as BSE prions in FVB mice, producing a prolonged and variable incubation period and a diglycosylated dominant PrPSc type (Hill et al., 1997) (Figures 2G, lane 2, and 4A; Table III). VCJD transmissions to SJL mice also resemble BSE transmissions to these mice. Inoculation with vCJD gives unusually short incubation times (Table III) and produces a monoglycosylated dominant PrPSc pattern which is closely similar to that produced by BSE transmission (Figures 2G, lane 5, and 4 A) and is unaffected by EDTA treatment (data not shown). To our knowledge, the PrPSc pattern seen on BSE or vCJD transmissions to RIIIS and SJL mice used in our study, or to the RJII strain of mice routinely used for biological strain typing experiments, has not been reported previously (Bruce et al., 1997). The neuropathological features seen in inbred lines of mice inoculated with the same prion strain vary considerably, the disease patterns being host, as well as prion strain, dependent (Bruce, 1993). The neuropathology observed in SJL and RIIIS mice inoculated with either BSE or vCJD showed only diffuse staining for PrP without florid or other PrP immunoreactive plaques (data not shown).

Discussion

Prion propagation involves recruitment and conversion of host PrPC into PrPSc, and the degree of primary structural similarity between inoculated PrPSc and host PrPC is thought to be a key component of intermammalian transmission barriers (Prusiner et al., 1990). It is clear, however, that prion strain type can also be crucial, as clearly demonstrated by the very distinctive transmission properties of sporadic CJD 129MM and vCJD 129MM prions (of identical PrP primary structure) in either 129VV Tgl52 (Hill et al., 1997; Collinge, 1999) or 129MM Tg35 mice. Prion strain type may also affect transmission barriers via an effect on PrPSc tertiary structure and state of aggregation (Hill et al, 1997; Collinge, 1999). These 129MM Tg35 mice, in which human PrPSc types can be propagated, have been used to study the BSE-to human species barrier. The frequent presence of subclinical prion disease in vCJD- and BSE- inoculated 129MM Tg35 mice further argues for the need to reassess current definitions of "species' or transmission barriers that limit prion transmission between different hosts (Hill et al, 2000). Such barriers have hitherto been quantitated on the basis of either comparative end-point titrations in the two respective hosts, or by measuring the fall in incubation period between primary and subsequent passage as the prion strain adapts to the new host. Both methods rely on measurement of time to onset of a clinical syndrome. Modelling the BSE-to-human barrier in 129MM Tg35 mice would lead to the conclusion, on the basis of induced clinical disease, that a substantial barrier existed. However, it is clear that human PrPSc propagation can be efficiently induced by inoculation with BSE or vCJD prions, suggesting a smaller barrier to infection (but not to clinical disease) than hitherto thought (Collinge et al., 1995) in humans of the PRNP 129MM genotype. Humans infected with BSE prions, but who became asymptomatic caniers, may nevertheless pose a threat of iatrogenic transmission via medical and surgical procedures. Alternatively, it is possible that the lifespan of the laboratory mouse is insufficient to allow expression of clinical disease in most inoculated mice, whereas a higher proportion of infected humans might survive the incubation period to develop clinical signs of disease. Serial passage studies and titration of prions in these mice are in progress to study this further. These studies further strengthen the evidence that vCJD is caused by a BSE-like prion strain. Also, remarkably, the key neuropathological hallmark of vCJD, the presence of abundant florid PrP plaques, can be recapitulated on BSE or vCJD transmission to these mice. However, the most surprising aspect of the studies was the finding that an alternate pattern of disease can be induced in 129MM Tg35 mice from primary transmission of BSE, with a molecular phenotype indistinguishable from that of a subtype of sporadic CJD. This finding has important potential implications as it raises the possibility that some humans infected with BSE prions may develop a clinical disease indistinguishable from classical CJD associated with type 2 PrPSc. This is, in our experience, the commonest molecular sub-type of sporadic CJD. In this regard, it is of interest that the reported incidence of sporadic CJD has risen in the UK since the 1970s (Cousens et al., 1997). This has been attributed to improved case ascertainment, particularly as much of the rise is reported from elderly patients and similar rises in incidence were noted in other European countries without reported BSE (Will et al., 1998). However, it is now clear that BSE is present in many European countries, albeit at a much lower incidence than was seen in the UK. While improved ascertainment is likely to be a major factor in this rise, that some of these additional cases may be related to BSE exposure cannot be ruled out. It is of interest in this regard that a 2-fold increase in the reported incidence of sporadic CJD in 2001 has recently been reported for Switzerland, a country that had the highest incidence of cattle BSE in continental Europe between 1990 and 2002 (Glatzel et al, 2002). No epidemiological case-control studies with stratification of CJD cases by molecular sub-type have yet been reported. It will be important to review the incidence of sporadic CJD associated with PrPSc type 2 and other molecular subtypes in both BSE-affected and unaffected countries in the light of these findings. If human BSE prion infection can result in propagation of type 2 PrPSc, it would be expected that such cases would be indistinguishable on clinical, pathological and molecular criteria from classical CJD. It may also be expected that such prions would behave biologically like those isolated from humans with sporadic CJD with type 2 PrPSc. The transmission properties of prions associated with type 2 PrPSc from BSE- inoculated 129MM Tg35 mice are being investigated by serial passage. We consider these data inconsistent with contamination of some of the 129MM Tg35 mice with sporadic CJD prions. These transmission studies were performed according to rigorous biosafety protocols for preparation of inocula and both the inoculation and care of mice, which are all uniquely identified by sub-cutaneous transponders. However, crucially, the same BSE inocula have been used on 129VV Tgl52 and 129MM Tg45 mice, which are highly sensitive to sporadic CJD but in which such transmissions producing type 2 PrPSc were not observed. Furthermore, in an independent experiment, separate inbred lines of wild-type mice, which are highly resistant to sporadic CJD prions, also propagated two distinctive PrPSc types on challenge with either BSE or vCJD. No evidence of spontaneous prion disease or PrPSc has been seen in groups of uninoculated or mock-inoculated aged 129MM Tg35 mice. While distinctive prion isolates have been derived from BSE passage in mice previously (designated 301C and 301V), these, in contrast to the data presented here, are propagated in mice expressing different prion proteins (Bruce et al., 1994). It is unclear whether our findings indicate the existence of more than one prion strain in individual cattle with BSE, with selection and preferential replication of distinct strains by different hosts, or that "mutation¹ of a unitary BSE strain occurs in some types of host. Western blot analysis of single BSE isolates has not shown evidence of the presence of a proportion of monoglycosylated dominant PrPSc type in addition to the diglycosylated dominant pattern (data not shown). Extensive strain typing of large numbers of individual BSE-infected cattle either by biological or molecular methods has not been reported. Presumably, the different genetic background of the different inbred mouse lines is crucial in determining which prion strain propagates on BSE inoculation. The transgenic mice described here have a mixed genetic background with contributions from FVB/N, C57BL/6 and 129Sv inbred lines; each mouse will therefore have a different genetic background. This may explain the differing response of individual 129MM Tg35 mice, and the difference between 129MM Tg35 and 129MM Tg45 mice, which are, like all transgenic lines, populations derived from single founders. Indeed, the consistent distinctive strain propagation in FVB and C57BL/6 versus SJL and RIIIS lines may allow mapping of genes relevant to strain selection and propagation, and these studies are in progress. That different prion strains can be consistently isolated in different inbred mouse lines challenged with BSE prions argues that other species exposed to BSE may develop prion diseases that are not recognizable as being caused by the BSE strain by either biological or molecular strain typing methods. As with 129MM Tg35 mice, the prions replicating in such transmissions may be indistinguishable from naturally occurring prion strains. It remains of considerable concern whether BSE has transmitted to, and is being maintained in, European sheep flocks. Given the diversity of sheep breeds affected by scrapie, it has to be considered that some sheep might have become infected with BSE, but propagated a distinctive strain type indistinguishable from those of natural sheep scrapie.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific prefened embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for canying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

TABLE 1

Table I. vCJD, BSE and sporadic CJD transmissions to transgenic mice expressing human PrP 129M

Inoculum Tg(HuPrP129M^+/+ Pmp⁰®)-^

Code PRNP129 Human PrP^Sc Clinical signs Incubation period Total genotype type (days ± SEM) affected⁸

Sporadic CJD 11199 MM Tl 3/3 237 ± 10 3/3

1120₂ MM Tl 8/8 229 ± 5 8/8

11196 MM Tl 8/8 22₅ ± 7 8/8

1026 MM T2 7/7 223 ± 1 7/7

1024 MV T2 4/4 241 ± 1 4/4

1022 VV T2 2/6 700, 708 4/6

1020 MV T3 6/7 437 ± 31 7/7

1021 VV T3 2/7 354 3 7 vCJD 1336 MM T4 0/2 >600 2/2

1342 MM T4 l/₅ 690 5/5

1344 MM T4 0 7 >340-720 7 7

BSE 103δ MM^b 2/20 344, 468 8/20

1060 MM^b 0/6 >570 1/6

1062 MM^b 2/7 338, 340 3/7

1064 M ¹" 2 10^c 344, 492 2/10

1066 MM¹" 0/6 >500 0/6

TABLE 11

Table 11. Summary of BSE and vCJD transmission to Tg3₅ and Tg45

Transgenic BSE vCJD

Total attack Clinical Sub-clinical Type 2 Type 4 Total attack Clinical Sub-clinical Type 2 Type 4 rate disease infection p-pSc p_rPSc rate disease infection rps- p-pSc

Tg35. 14/49 6/49 8/49 10/11" 1/1 l^a 14/14 1/14 13/14 0/14 14/14 Tg45 9/12 0/12 9/12 0/9 9/9 4/4 1/4 3/4 0/4 4/4

"Three brains not analysed by western blotting (one brain from a clinically infected animal was unavailable for either western blotting or immunohistochemistry; single brains from clinically affected and sub-clinically affected animals were scored positive by immunohistochemistry).

TABLE III

Table HI. BSE and vCJD transmissions to inbred lines of mice

Inoculum SJ /OlaHsd RIIIS/J FVB/NHsd C57B /6/θlaHsd

Incubation Clinical Incubation Clinical Incubation Clinical Incubation Clinical time signs time signs time signs time signs

(days ± SEM) (days ± SEM) (days ± SEM) (days ± SEM)

BSE (1783) 196 ± 13 25/40 241 ± 15 20/29 589 ± 21 22/31 710 ± 15 6/25 vCJD (1336) 139 ± 17 6/10 342 ± 31 8/8 vCJD (1344) 256 ± 46 5/7 402 ± 34 7/8 vCJD (1342) 169, 169 2/11 475 ± 68 3/10

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Claims

1. A transgenic non-human animal expressing, as a result of transgene expression, a PrP gene homozygous for M129, wherein contacting said transgenic non-human animal with a sample containing prions causes prion infection that is characteristic of human prion infection.

2. A transgenic non-human animal according to claim 1 wherein the PrP gene homozygous for Ml 29 is a human PrP gene.

3. A transgenic non-human animal according to claim 1 or claim 2 wherein the prions contacted with the transgenic non-human animal cause prion infection in humans or bovines.

4. A transgenic non-human animal according to claim 3 wherein the prions cause sCJD, vCJD or BSE in their natural hosts.

5. A transgenic non-human animal according to claim 3 or claim 4 wherein the prions have aPENP129MM genotype.

6. A transgenic non-human animal according to any one of the preceding claims wherein the human prion infection is characteristic of vCJD.

7. A transgenic non-human animal according to any one of the preceding claims wherein the transgenic non-human animal is a mammal.

8. A transgenic non-human animal according to claim 7 wherein the mammal is a mouse.

9. A method for identifying one or more agents capable of modulating human prion infection, comprising the steps of: a) contacting a transgenic non-human animal expressing, as a result of transgene expression, a PrP gene homozygous for M129, with a sample comprising one or more prions;

b) contacting the transgenic non-human animal with one or more agents;

c) determining the effect of the agents on prion infection; and

d) selecting one or more agents which are capable of modulating prion infection.

10. A method according to claim 9 wherein the PrP gene homozygous for M129 is a human PrP gene.

11. A transgenic non-human animal according to claim 9 or claim 10 wherein the prions contacted with the transgenic non-human animal cause prion infection in humans or bovines.

12. A transgenic non-human animal according to claim 11 wherein the prions cause sCJD, vCJD or BSE in their natural hosts.

13. A transgenic non-human animal according to claim 11 or claim 12 wherein the prions have aPRNP129MM genotype.

14. A method according to any one of claims 9 to 13 wherein the prion infection is characteristic of vCJD.

15. A method according to any one of claims 9 to 14 wherein the transgenic non- human animal is a mammal.

16. A method according to claim 15 wherein the mammal is a mouse.

17. A process comprising the steps of:

i) performing the method according to any one of claims 9 to 16; ii) identifying an agent capable of modulating human prion infection; and

iii) preparing a quantity of that agent.

18. A process comprising the steps of:

i) performing the method according to any one of claims 9 to 16; ii) identifying an agent capable of modulating human prion infection; iii) preparing a quantity of that agent; and iv) preparing a pharmaceutical composition comprising that agent.

19. A process comprising the steps of:

i) performing the method according to any one of claims 9 to 16; ii) identifying an agent capable of modulating prion infection; iii) modifying said agent; and iv) preparing a pharmaceutical composition comprising said modified agent.

20. A pharmaceutical composition comprising an agent identified by the assay method of any one of claims 9 to 16 or the process of any one of claims 17 to 19 admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant and or combinations thereof.

21. A process of preparing a pharmaceutical composition comprising admixing an agent identified by the assay method of any one of claims 9 to 16 or the process of any one of claims 17 to 19 with a pharmaceutically acceptable diluent, carrier, excipient or adjuvant and/or combinations thereof.

22. A method of treating human prion infection which method comprises administering to an individual an effective amount of a pharmaceutical composition comprising an agent identified by the assay method of any one of claims 9 to 16 or the process of any one of claims 17 to 19, wherein the agent is capable of modulating prion infection and wherein said composition is optionally admixed with a pharmaceutically acceptable carrier, diluent excipient or adjuvant and/or combinations thereof.

23. A method according to claim 22 wherein said one or more agents are formulated into one or more compositions for use in medicine.

24. An agent identifiable, preferably identified by the assay method according to any one of claims 9 to 16.

25. An agent identifiable preferably identified by the assay method according to any one of claims 9 to 16 for use in the treatment and/or prevention of disease.

26. Use of an agent identifiable, preferably identified by the assay method according to any one of claims 9 to 16 in the manufacture of a composition for the treatment and/or prevention of human prion infection.

27. Use of a transgenic non-human animal according to any one of claims 1 to 8 as a model for human prion infection.