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Detection of conformationally altered proteins and prions

This application is a continuation of U.S. patent application Ser. No. 10/728,246 filed Dec. 4, 2003, which is a continuation-in-part of U.S. patent application Ser. No. 10/161,061, filed May 30, 2002, which claims priority from U.S. Provisional Patent Application Ser. No. 60/295,456, filed May 31, 2001. This application is also a continuation-in-part of U.S. application Ser. No. 10/494,906 filed Sep. 7, 2004, which is a National Stage of Application Serial No. PCT/US02/17212 filed May 30, 2002, which claims priority from U.S. Provisional Application Ser. No. 60/295,456 filed May 31, 2001. The entire contents of the aforementioned applications are incorporated herein by reference.

The invention provides methods and kits for detecting conformationally altered proteins and prions in a sample.

In one embodiment, the conformationally altered proteins and prions are associated with amyloidogenic diseases.

The conversion of normally soluble proteins into conformationally altered insoluble proteins is thought to be a causative process in a variety of other diseases. Structural conformational changes are required for the conversion of a normally soluble and functional protein into a defined, insoluble state. Examples of such insoluble proteins include: A. beta. peptide in amyloid plaques of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA); .alpha.-synuclein deposits in Lewy bodies of Parkinson's disease, tau in neurofibrillary tangles in frontal temporal dementia and Pick's disease; superoxide dismutase in amylotrophic lateral sclerosis; huntingtin in Huntington's disease; and prions in Creutzfeldt-Jakob disease (CJD): (for reviews, see Glenner et al. (1989) J. Neurol. Sic. 94; 1-28; Haan et al. (199) Clin. Neurol. Neurosurg. 92(4):305-310).

Often these highly insoluble proteins form aggregates composed of nonbranching fibrils with the common characteristic of a beta.-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur (1989) Acta Neuropathol. 78:329-331; Kawai et al. (1993) Brain Res. 623:142-6; Martin et al. (1994) Am. J. Pathol. 145:1348-1381; Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996) J. Neurosci. 16:5795-5811). Other studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996) Nat. Med. 2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy (1997) Trends Neurosci. 20:154-9).

In both AD and CAA, the main amyloid component is the amyloid beta protein (A. beta.). The A. beta. peptide, which is generated from the amyloid beta precursor protein (APP) by the action of two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, A.beta1-40 and A.beta1-42, are produced by alternative carboxy-terminal truncation of APP (Selkoe et al. (1988) Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409). A.beta1-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy (1997), supra; Haan et al. (1990), supra; Terry et al., supra; Vinters (1987), supra; Itoh et al. (1993), supra; Yamada et al. (1993), supra; Greenberg et al. (1993), supra; Levy et al. (1990), supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.

Human transthyretin (TTR) is a normal plasma protein composed of four identical, predominantly beta.-sheet structured units, and serves as a transporter of the hormone thyroxin. Abnormal self assembly of TTR into amyloid fibrils causes two forms of human diseases, namely senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) (Kelly (1996) Curr Opin Struct Biol 6(1):11-7). The cause of amyloid formation in FAP is point mutations in the TTR gene; the cause of SSA is unknown. The clinical diagnosis is established histologically by detecting deposits of amyloid in situ in biopsy material.

To date, little is known about the mechanism of TTR conversion into amyloid in vivo. However, several laboratories have demonstrated that amyloid conversion may be simulated in vitro by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993) Biochemistry 32(45):12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun 197(2) 415-21]. The mechanism of conformational transition involves a monomeric conformational intermediate which poly_merizes into linear beta.-sheet structured amyloid fibrils [Lai, Colon et al. (1996) Biochemistry 35(20):6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxin or triiodophenol (Miroy, Lai et al. (1996) Proc Natl Acad Sci USA 93(26):15051-6).

The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the disease-associated neurodegenerative processes are not well-defined. The amyloid fibrils in the brains of Alzheimer's and prion disease patients are known to result in the inflammatory activation of certain cells. For example, primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar .beta.-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The signaling response elicited by .beta.-amyloid and prion fibrils leads to the production of neurotoxic products, which are in part responsible for the neurodegeneration. C. K. Combs et al, J Neurosci 19:928-39 (1999).

Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. A potential prion precursor is a protein referred to as PrP 27-30, a 28 kdalton hydrophobic glycoprotein that poly_merizes (aggregates) into rod-like filaments found as plaques in infected brains. The normal protein homologue differs from prions in that it is readily degradable, whereas prions are highly resistant to proteases. It has been suggested that prions may contain extremely small amounts of highly infectious nucleic acid, undetectable by conventional assay methods Benjamin Lewin, Genes IV (Oxford Univ. Press, New York, 1990 at p. 1080. The predominant hypothesis at present is that no nucleic acid component is necessary for the infectivity of prion protein.

Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPC is encoded by a single-copy host gene and is normally found at the outer surface of neurons. During a post-translational process, PrPSc is formed from the normal, cellular PrP isoform (PrPC), and prion diseases result from conversion of PrPC into a modified isoform called PrPSc. PrPSc is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans.

The normal prion protein (PrP) is a cell-surface metallo-glycoprotein that is mostly an alpha-helix and coiled-loop structure as shown in FIG. 8, and is usually expressed in the central nervous and lymph systems. It is believed to serve as an antioxidant and is thought to be associated with cellular homeostasis. The abnormal form of PrP, however, is a confor_mer which is resistant to proteases and is predominantly beta-sheet in its secondary structure, as shown in FIG. 9. It is believed that this conformational change in secondary structure leads to aggregation and eventual neurotoxic plaque deposition in the prion-disease process.

Prion-associated diseases include scrapie of sheep and goats, chronic wasting disease of deer and elk, and bovine spongiform encephalopathy (BSE) of cattle (Wilesmith, J. and Wells, Microbiol. Immunol. 172:21-38 (1991)). Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) (Gajdusek, D. C., Science 197:943-960 (1977); Medori et al., N. Engl. J. Med. 326:444-449 (1992)).

Prion diseases are transmissible and insidious. For example, the long incubation times associated with prion diseases will not reveal the full extent of iatrogenic CJD for decades in thousands of people treated with cadaver-sourced HGH worldwide. The importance of detecting prions in biological products has been heightened by the possibility that bovine prions have been transmitted to humans who developed new variant Creutzfeldt-Jakob disease (nvCJD) (G. Chazot et al., Lancet 347:1181 (1996); R. G. Will et al. Lancet 347:921-925 (1996)).

Diseases caused by prions are hard to diagnose: the disease may be latent or subclinical (abnormal prions are detectable but symptoms are not). Moreover, normal homologues of a prion-associated protein exist in the brains of uninfected organisms, further complicating detection. Ivan Roitt, et al., Immunology (Mosby-Year Book Europe Limited, 1993), at 15.1.

Current techniques used to detect the presence of prion-related infections rely on gross morphological changes in the brain and immunochemical techniques that are generally applied only after symptoms are manifest. Many of the current detection methods rely on antibody-based assays or affinity chromatography that use brain tissue from dead animals, and in some cases capillary immunoelectrophoresis of blood samples.