Transgenic animals lacking prion proteins

Details Transgenic animals lacking prion proteins Transgenic animals lacking prion proteins

1994-06-21

1997-12-16

Ziska, Suzanne E.

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

536 231, 536 235, 4351723, 4352402, 800DIG1, C12N 1500, C12N 500, C07H 2104

Patent

active

056987633

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD OF THE INVENTION

This invention relates to transgenic mammals and birds ("animals") that are not susceptible to spongiform encephalopathies (i.e., scrapie-like or prion diseases), due to absence of endogenous prion protein ("PrP"). More particularly, this invention relates to targeting molecules that are capable of disrupting PrP genes, PrP genes that are disrupted by such targeting molecules and thus incapable of expressing functional prion protein, to cultured cells transformed to have such disrupted genes, to mammals and birds derived from those transformed cells and to the transgenic progeny of such animals.


BACKGROUND OF THE INVENTION

Prions are infectious pathogens that cause 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.
Kuru, Creutzfeldt-Jakob disease ("CJD") and Gerstmann-Straussler-Scheinker syndrome ("GSS") are fatal human neurodegenerative diseases caused by prions. Scrapie of sheep and goats is the most well-studied prion disease. Bovine spongiform encephalopathy ("BSE" or "mad cow disease") is a prion disease that currently threatens the beef industry in Great Britain. Transmissible mink encephalopathy and chronic wasting disease of deer and elk are also thought to be prion diseases. The pathological characteristics of prion diseases include neuronal vacuolation, astrocytic gliosis, and amyloid plaques with filaments composed of prion protein.
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., Science 218, pp. 1309-11 (1982); Prusiner et al., Biochemistry 21, pp. 6942-50 (1982); McKinley et al., Cell 35, pp. 57-62 (1983)). Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrP.sup.C is encoded by a single-copy host gene (Basler et al., Cell 46, pp. 417-28 (1986)) and is normally found at the outer surface of neurons. A leading hypothesis is that prion diseases result from conversion of PrP.sup.C into a modified form called PrP.sup.Sc.
The actual biological or physiological function of PrP.sup.C is not known. Suggestions that it is identical with acetylcholine receptor inducing activity ("ARIA") remain to be confirmed (Harris et al., Proc. Natl. Acad. Sci. USA 88, pp. 7664-68 (1991)). The PrP gene, however, is found in all mammals so far examined, i.e., hamster (Basler et al., Cell 46, pp. 417-28 (1986)), mouse (Locht et al., Proc. Natl. Acad. Sci. USA 83, pp. 6372-76 (1986)), human (Kretzschmar et al., DNA 5, 315-24 (1986); Puckett et al., Am. J. Hum. Genet. 49, 320-29 (1991)), rat (Westaway and Prusiner, Nucl. Acids Res. 14, 2035-44 (1986)), cattle (Goldmann et al., J. Gen. Virol. 72 pp. 201-04 (1991)), sheep (Goldmann et al., Proc. Natl. Acad. Sci USA 87, pp. 2476-80 (1990)), and goat (Westaway and Prusiner, supra). The PrP gene is also found in chickens (Harris et al., Proc. Natl. Acad. Sci. USA 88, pp. 7664-68 (1991)). Furthermore, because PrP.sup.C is expressed in neurons of the brain (Kretzschmar et al., Am. J. Pathol. 122, pp. 1-5 (1986)) as well as in lymphocytes (Cashman et al., Cell 61, pp. 185-92 (1990)) and shows a high turnover rate (Caughey et al., J. Virol 63, pp. 175-81 (1989); Borchelt et al., J. Cell. Biol. 110, pp. 743-52 (1990)), it would seem to be of considerable physiological importance.
The central role of PrP.sup.C in scrapie-like diseases is indicated by several lines of evidence. The infectious agent copurifies with PrP.sup.Sc by different procedures (Prusiner, Science 216, pp. 136-44 (1982); Diringer et al., Nature 306, pp. 476-78 (1983); McKinley et al., Cell 35, pp. 57-62 (1983); Hope et al., EMBO J. 5, pp. 2591-97 (1986)), including affinity purification on an anti-PrP antibody column (Gabizon et al., Proc. Natl. Acad. Sci. USA 85, pp. 6617-21 (1988)), SDS-polyacrylamide gel electrophoresis (Brown

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