Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-09-25
2003-08-05
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S069100, C435S320100, C435S325000, C435S252300, C536S023100, C530S350000
Reexamination Certificate
active
06602672
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to epitope-tagged proteins and to transgenic animals expressing such proteins. More specifically, this invention relates to epitope-tagged prion protein (PrP) genes, transgenic animals expressing epitope-tagged PrP genes, and assay.methods for distinguishing between and isolating infectious and noninfectious prion proteins.
BACKGROUND OF THE INVENTION
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not infect another (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (“PrP”) (Bolton et al. (1982) Science 218:1309-11; Prusiner et al. (1982) Biochemistry 21:6942-50; McKinley et al. (1983) Cell 35:57-62). Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrP
C
is encoded by a single-copy host gene (Basler et al. (1986) Cell 46:417-28) and is normally found at the outer surface of neurons. A leading hypothesis is that prion diseases result from conversion of PrP
C
into a modified scrapie isoform called PrP
Sc
during a post-translational process (Borchelt et al. (1990) J. Cell Biol. 110:743-752). It is likely that a fundamental event in the propagation of prions is the conformational transition of alpha-helices in PrP
C
into beta-sheets in PrP
Sc
(Pan et al. (1993) Proc. Natl. Acad. Sci. 90:10962-10966). Genetic evidence from transgenic mouse studies demonstrates the requirement for an additional component(s) referred to as protein X in this conversion (Telling et al. (1995) Cell 83:79-90).
It appears that PrP
Sc
is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans (see, Prusiner (1991) Science 252:1515-1522). The most common prion diseases of animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle (Wilesmith & Wells (1991) Microbiol. Immunol. 172:21-38). 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 (1977) Science 197:943-960; Medori et al. (1992) N. Engl. J. Med. 326:444-449). The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
Most CJD cases are sporadic, but about 10-15% are inherited as autosomal dominant disorders that are caused by mutations in the human PrP gene (Hsiao et al. (1990) Neurology 40:1820-1827; Goldfarb et al. (1992) Science 258:806-808); Kitamoto et al. (1994)
Proc. R. Soc. Lond
. 343:391-398). Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts (Brown et al. (1992) Lancet 340:24-27). attempts to link CJD to an infectious source such as the consumption of scrapie infected sheep meat, none has been identified to date (Harries-Jones et al. (1988) J. Neurol. Neurosurg. Psychiatry 51:1113-1119) except in cases of iatrogenically induced disease. On the other hand, kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism (Alpers (1979)
Slow Transmissible Diseases of the Nervous System
, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 66-90).
The initial transmission of CJD to experimental primates has a rich history beginning with William Hadlow's recognition of the similarity between kuru and scrapie. In 1959, Hadlow suggested that extracts prepared from patients dying of kuru be inoculated into non-human primates and that the animals be observed for disease that was predicted to occur after a prolonged incubation period (Hadlow (1959) Lancet 2:289-290). Seven years later, Gajdusek, Gibbs and Alpers demonstrated the transmissibility of kuru to chimpanzees after incubation periods ranging from 18 to 21 months (Gajdusek et al. (1966) Nature 209:794-796). The similarity of the neuropathology of kuru with that of CJD (Klatzo et al. (1959) Lab Invest. 8:799-847) prompted similar experiments with chimpanzees and transmissions of disease were reported in 1968 (Gibbs, Jr. et al. (1968) Science 161:388-389). Over the last 25 years, about 300 cases of CJD, kuru and GSS have been transmitted to a variety of apes and monkeys.
The expense, scarcity and often perceived inhumanity of such experiments have restricted this work and thus limited the accumulation of knowledge. While the most reliable transmission data has been said to emanate from studies using non-human primates, some cases of human prion disease have been transmitted to rodents but apparently with less regularity (Gibbs, Jr. et al. (1979)
Slow Transmissible Diseases of the Nervous System
, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 87-110; Tateishi et al. (1992)
Prion Diseases of Humans and Animals
, Prusiner et al., eds. (London: Ellis Horwood), pp. 129-134).
The infrequent transmission of human prion disease to rodents has been cited as an example of the “species barrier” first described by Pattison in his studies of passaging the scrapie agent between sheep and rodents (Pattison (1965)
NINDB Monograph
2, D. C. Gajdusek, C. J. Gibbs Jr. and M. P. Alpers, eds. (Washington, D.C.: U.S. Government Printing), pp. 249-257). In those investigations, the initial passage of prions from one species to another was associated with a prolonged incubation time with only a few animals developing illness. Subsequent passage in the same species was characterized by all the animals becoming ill after greatly shortened incubation times.
The molecular basis for the species barrier between Syrian hamster (SHa) and mouse (Mo) was shown to reside in the species-specific differences in the sequence of the PrP (Scott et al. (1989) Cell 59:847-857). Mouse PrP (MoPrP) differs from Syrian hamster PrP (SHaPrP) at 16 positions out of 254 amino acid residues (Basler et al. (1986), Cell supra; Locht et al. (1986) Proc. Natl. Acad. Sci. USA 83:6372-6376). Transgenic mice expressing SHaPrP [Tg(SHaPrP)] had abbreviated incubation times when inoculated with SHa prions. When similar studies were performed with mice expressing the human, or ovine PrP transgenes, the species barrier was not abrogated, i.e., the percentage of animals which became infected were unacceptably low and the incubation times were unacceptably long (Telling et al. (1994) Proc. Natl. Acad. Sci. 91:9936-9940; Telling et al. (1995) Cell 83:79-90). Thus, it was not possible to render non-human animals such as mice, susceptible to infection by human prions.
Purification of PrP
Sc
has been facilitated by its relative resistance to proteolytic degradation and insolubility in non-denaturing detergents (Bolton et al. (1982) supra; Prusiner et al. (1982) supra. Purification of PrP
C
has been more problematic. Immunoaffinity chromatography purification of PrP
C
yielded only small amounts of protein. Improved purification of PrP
C
has been accomplished by a multi-step purification procedure involving detergent extraction and separation by immobilized Cu
2+
ion affinity chromatography followed by cation-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Pan et al. (1992) Protein Sci. 1:136-144).
The production of monoclonal antibodies against PrP
C
and PrP
Sc
has been particularly difficult. In the case of mouse PrP, MoPrP is recognized as self, precluding the production of anti-MoPrP antibodies in animals immunized with MoPrP.
There is an urgent ne
Cohen Fred E.
Prusiner Stanley B.
Scott Michael R.
Telling Glenn C.
Bozicevic Karl
Bozicevic Field & Francis LLP
Carlson Karen Cochrane
The Regents of the University of California
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