This invention relates to molecules (for example, peptides) that bind selectively to the disease-specific abnormal isoform of the prion protein, herein generically designated PrPSc, and not to the normal isoform of this protein, designated PrPC. These molecules are useful for detecting PrPSc in a sample, and for purifying PrPSc. Additionally, the invention relates to diagnostic aids for the detection of PrPSc, pharmaceuticals that inhibit the recruitment of normal PrPC to the disease-specific PrPSc, and methods for prion decontamination.
The prion diseases are a group of rapidly progressive, fatal, and untreatable neurodegenerative syndromes. Human prion diseases include classical Creutzfeldt-Jakob disease (CJD), which has sporadic, iatrogenic, and familial forms. More recently, a variant CJD (vCJD) has been recognized in the United Kingdom, France, the Republic of Ireland, Hong Kong, Italy and the United States (Will et al., Lancet 347:921-25, 1996; Collinge, Lancet 354:317-23, 1999), likely derived from the consumption of cattle tissues contaminated with the agent of bovine spongiform encephalopathy (“BSE”; reviewed in Cashman, Can. Med. Assoc. J. 157:1381-5, 1997; Coulthart and Cashman, Can. Med. Assoc. J. 165:51-8, 2001). More than 173,000 cattle, primarily from Britain, have developed symptomatic BSE, and many thousands more have probably entered the food supply undetected. The primary epidemic of vCJD has been predicted to range from dozens to hundreds of thousands of afflicted individuals, based on various poorly understood assumptions, such as the incubation period of the disease. Moreover, classical CJD has been accidentally transmitted between humans by contaminated cadaveric pituitary hormones, dura mater transplantation, neurosurgical instrumentation, and corneal transplantation (Brown et al., Neurology 55:1075-8, 2000). A “secondary epidemic” of vCJD through blood and blood products, general surgery, dentistry, vaccines, and cosmetics cannot be ruled out at present. The detection of blood prion infectivity in experimental BSE/vCJD infections of mice and sheep (Taylor et al., J. Hosp. Infect. 46:78-9, 2000; Houston et al., Lancet 356:999-1000, 2000), but not in classical CJD (Rickets et al., Emerg. Infect. Dis. 3:155-63,1997) suggests a special risk of transmitting vCJD through blood and blood products. The United States and Canada have now implemented a blood donor deferral for individuals who resided in the UK during the BSE epidemic, and Canada has also deferred donors from France. Donor deferrals extended to Western Europe are now being implemented in the United States and Canada.
Prions are the infectious agents that are associated with the transmissible spongiform encephalopathies noted above. The prion diseases are neurodegenerative syndromes characterized by spongiform change (e.g., microcavitation of the brain, usually predominant in gray matter), neuronal cell loss, astrocytic proliferation disproportionate to neuronal loss, and accumulation of an abnormal amyloidogenic protein, sometimes in discrete plaques in the brain. The agents that transmit these diseases differ markedly from viruses and viroids in that no chemical or physical evidence for a nucleic acid component has been reproducibly detected in infectious materials (Prusiner, Science 216: 136-144, 1982). A major step in the study of model scrapie prions was the discovery and purification of a protein designated the scrapie-associated prion protein (PrPSc) (Bolton et al., Science 218:1309-11, 1982; Prusiner et al., Biochemistry 21:6942-50, 1982; McKinley et al., Cell 35:57-62, 1983). When purified using proteinase K digestion, a 27-30 kD protease-resistant protein was discovered in scrapie-affected hamster brain, and was termed PrP 27-30, later found to be a fragment of PrPSc (Bolton et al., Science 218:1309-1311, 1982).
According to the prion hypothesis, prion infectivity resides in PrPSc, or a related conformational intermediate. PrPSc is the most prominent (or perhaps sole) macromolecule in preparations of prion infectivity, and appears to be at least a reliable surrogate for most prion infection. PrPSc is a conformational variant of a host-encoded cellular protein designated PrPC (Oesch et al., Cell 40:735-746, 1985), which is a glycosylphosphatidylinositol (GPI)-linked cell surface protein with a molecular mass of 33-35 kD. PrPC has been isolated from normal brain, and has been found to be protease-sensitive and not associated with scrapie disease-producing activity (Bolton and Bendheim Ciba Found. Symp. 135:164-177, 1988). According to the prion theory, PrPC converts into PrPSc in a template-directed process initiated by contact with PrPSc (Prusiner, Proc. Natl. Acad. Sci., U.S.A. 95:13363-83, 1998).
PrPC is an evolutionarily conserved membrane protein for which the actual biological or physiological function is unclear. Mice devoid of PrPC are viable and show no obvious signs of neurological and physical impairment (Bueler et al., Nature 356:577-582, 1992), except for an ataxic syndrome in certain PrP knockout mouse strains due to upregulation in brain of the prion homolog protein dopple (Moore et al., J Mol. Biol. 292:797-817, 1999). Prnp knockout mice are not susceptible to prion infection, underscoring the central importance of PrPC in the replication of infectivity (Bueler et al., Cell 73:1339-1347, 1993; Prusiner et al., Proc. Natl. Acad. Sci., U.S.A. 90:10608-10612, 1993). Targeted investigations of Prnp knockout mice revealed impaired synaptic function (Collinge et al., Nature 370:295-297, 1994) and altered sleep regulation (Tobler et al., J. Neurosci. 17:1869-79, 1997). Moreover, antibody-mediated ligation of PrPC at the cell surface has been shown to depress T cell activation (Cashman et al., Cell 61:185-192, 1990; Li et al., Cell. Immunol. 207:49-58 2001), suggesting a role for the protein in immune function.
PrPC is present in large excess to PrPSc in accessible peripheral tissues and organs of animals and humans afflicted with prion diseases. The availability of reagents that distinguish PrPSc from PrPC would therefore be of great value in development of a test for prion infection in blood or other tissues accessible to sampling. Furthermore, in view of the epidemic nature of BSE and vCJD, a pressing need exists for therapeutic agents that prevent and/or treat prion diseases. Accordingly, a need exists in the art for testing samples for the presence of prions, methods for treating prion diseases, as well as for developing anti-prion pharmaceuticals and decontaminants.
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USPTO Backgrounds