Contributed by Stanley B. Prusiner, August 24, 1993. ABSTRACT. Mice, homozygous ..... irradiation studies and Michael Baldwin for reviewing the manu- script.
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10608-10612, November 1993
Medical Sciences
Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies (immune tolerance/gene targetting/prion diseases/gene therapy/antisense prion protein)
STANLEY B. PRUSINER*t*, DARLENE GROTH*, ANA SERBAN*, RUTH KOEHLER*, DALLAS FOSTER*, MARILYN TORCHIA*, DENNIS BURTON§, SHU-LIAN YANG*, AND STEPHEN J. DEARMOND1 Departments of *Neurology, tBiochemistry and Biophysics, and lPathology, University of California, San Francisco, CA 94143; and §The Scripps Research Institute, La Jolla, CA 92037
Contributed by Stanley B. Prusiner, August 24, 1993
ABSTRACT Mice, homozygous for prion protein (PrP) gene ablation (Prn-p°/°), develop normally and remain well >500 days after inoculation with murine scrapie prions. In contrast, wild-type mice developed scrapie 175 >525 10 156 ± 5.6 169 ± 13 PMn-p+/+ 9 512 ± 10 530 ± 13 9 18 >500 151 2.0 165 ± 3.4 CD-1 9 >450 10 >525 9 430 ± 21 426 ± 18 Prn-p+/° 8 10 >450 8 >450 >510 Prn-p°/° 42 ± 3.0 11 26 40 ± 3.0 25 48 ± 1.0 51 ± 1.0 181 ± 16 174 ± 17 Tg(SHaPrP+/0)7 6 42 ± 0 40 ± 0 50 ± 1.6 7 49 ± 1.5 Tg(SHaPrP+/+)7 26 112 ± 9.5 117 ± 8.0 22 75 ± 1.1 75 ± 1.1 20 200 ± 3.2 194 ± 3.5 Tg(SHaPrP+/0)81 10 58 ± 1.1 58 ± 1.1 60 ± 2.9 10 12 57 ± 2.2 201 ± 5.0 198 ± 5.4 Tg(SHaPrP+/+)81 9 54 ± 1.1 56 ± 1.8 10 >150 Tg(SHaPrP+/0)81/Prn-p0/° *Incubation time in days (means ± SE). tNumber of animals with scrapie or well after a specified period of time. In some experiments, fewer animals were used to calculate the death times because a fraction of the ill animals were sacrificed for neuropathological studies.
Medical Sciences: Prusiner et al. 1
SHa Mo Hu 2 3 4 5 6 7 8 9
Proc. Natl. Acad. Sci. USA 90 (1993)
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compared to Tg(SHaPrP+/0)81/Prn-p+/+ mice with times of -75 days. Interestingly, Tg(SHaPrP+/+)81/Prn-p+/+ mice also had -50-day incubation times. To date, we have encountered difficulty in producing Tg(SHaPrP+/+)81/Pm-
polo
FIG. 3. Western immunoblots demonstrating anti-PrP Abs produced in Pmn-p0!0 mice after immunization with Mo prions. Lanes: 1-3, Syrian hamster brain; 4-6, CD-i mouse; 7-9, human; 1, 4, and 7, normal homogenates; 2 and 5, scrapie-infected homogenates; 3 and 6, purified scrapie prions containing NrP 27-30; 8, homogenate from a patient dying of sporadic Creutzfeldt-Jakob disease (CJD); 9, purified CJD prions containing PNP 27-30. Molecular size values for proteins are denoted by horizontal lines and were determined by using prestained markers: phosphorylase b, 106 kDa; bovine serum albumin, 80 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa; lysozyme, 18.5 kDa.
Tg(SHaPrP+/0)81/Prn-p0/0 mice inoculated with SHa(Sc237) prions. Incubation times of -150 days after inoculation with Mo(RML) prions in Pr-p+/+ and CD-i Swiss mice with two NrP alleles contrast to longer times in Tg(SHaPrP) mice expressing high levels of transgene SHaPrPC (17) and in PMn-Po/+ mice expressing low levels of MoNPPC (15). Production of Anti-NrP Abs. We immunized Prn-p0/0 mice with Mo and SHa prions dispersed in Freund's adjuvant. In our initial study, 1 of 2 Nrn-p0/0 mice immunized with Mo prions and 0 of 2 mice immunized with SHa prions produced anti-NrP Abs. In a second study, 10 of 10 mice immunized with Mo prions and 10 of 10 mice immunized with SHa prions produced anti-NrP Abs. Anti-NrP Abs in sera were detected by Western blotting. The anti-NrP Abs reacted with Mo, SHa, and human NP (Fig. 3). The anti-NP antisera had titers exceeding 1: 5000. DISCUSSION Although there has been a remarkable convergence of experimental results contending that rnPsc is essential for transmission and pathogenesis of prion diseases (6), this conclusion continues to be challenged by some investigators (44-48). The inoculation of Pmn-p00 mice with scrapie prions provided yet another opportunity to test the hypothesis that NPpsc is neither required for development of disease nor necessary for multiplication of scrapie infectivity. If Pm-p0i 0 mice inoculated with Mo prions had developed disease or propagated infectivity, then it could be argued that Psc is unrelated to the transmis'sion as well as the pathogenesis of disease or that Mo prions stimulate the conversion of another cellular protein into a pathological isoform through an autocatalytic process. Since Pm-p0S0mice are resistant to scrapie and fail to propagate scrapie infectivity as presented here and elsewhere (20), we conclude that the PrP is necessary for both disease pathogenesis and prion propagation. Increased SHaPrP transgene expression shortened incubation times for homologous SHa prions and extended them for heterologous Mo prions (16, 17, 19). Crosses between Tg(SHaPrP/)81 and Pmn-p00 mice inoculated with SHa prions (Table 2) lend further support to the hypothesis that competition between endogenous MoPrPC and transgene PrPc for inoculated PrPsc modulate incubation times. The Tg(SHaPrP+/0)81/Prn-p0/0 mice heterozygous for the SHaPrP transgene array had incubation times of -50 days
Whether conformation-dependent mAbs that discriminate between PrPSc and PrPC can be produced from Prn-p°/0 mice producing anti-MoPrP Abs (Fig. 3) remains to be determined but such mAbs would be invaluable. Spectroscopic data show that prPC has a high a-helical content and virtually no ,&-sheet (37), supporting a four-helix bundle model for PrPC (49). Since PrPSc has a high /-sheet content, it is likely that conversion of prPC into PrPsc involves the unfolding of a-helical domains and their refolding into /-sheets (37). Since ablation of both alleles of the PrP gene does not seem to be deleterious to mice, scrapie cannot be considered a disease of PrPC inhibition (15). Rather, considerable evidence argues that the accumulation of PrPsc is responsible for the central nervous system dysfunction that attends scrapie (43, 50-52). In accord with these findings is the slow deposition of PrPSc in Prn-pO/+ mouse brains, which is accompanied by a prolongation of the incubation time (Figs. 1 and 2). The results presented here and by others (20) with Prn-p0/0 mice suggest therapeutics for prion diseases. Gene-targeted domestic animals such as sheep and cattle could be produced with ablated PrP genes if this does not prove deleterious to the health of the animals. Such sheep would be protected from scrapie and the cattle would be protected from bovine spongiform encephalopathy (53). Alternatively, antisense PrP genes or oligonucleotides should reduce PrP mRNA levels and thus diminish the production of PrPsc. Whether such therapy might be effective in humans who present early in the clinical course of a prion disease is unknown, but it could be administered to patients without symptoms who are at risk for inherited prion diseases. We thank Charles Weissmann for the Prn-p0/0 mice and his stimulating discussions. We thank James Cleaver for help with irradiation studies and Michael Baldwin for reviewing the manuscript. This research was supported by research grants from the National Institutes of Health and the American Health Assistance Foundation as well as by gifts from the Sherman Fairchild Foundation and National Medical Enterprises. 1. Chandler, R. L. (1961) Lancet i, 1378-1379. 2. Parry, H. B. (1983) in Scrapie Disease in Sheep, ed. Oppenheimer, D. R. (Academic, New York). 3. Dickinson, A. G., Young, G. B., Stamp, J. T. & Renwick, C. C. (1%5) Heredity 20, 485-503. 4. Parry, H. B. (1962) Heredity 17, 75-105. 5. Prusiner, S. B. (1989) Annu. Rev. Microbiol. 43, 345-374. 6. Prusiner, S. B. (1991) Science 252, 1515-1522. 7. Prusiner, S. B., Hadlow, W. J., Garfin, D. E., Cochran, S. P., Baringer, J. R., Race, R. E. & Eklund, C. M. (1978) Biochemistry 17, 4993-4997. 8. Prusiner, S. B., Garfin, D. E., Cochran, S. P., McKinley, M. P. & Groth, D. F. (1980) J. Neurochem. 35, 574-582. 9. Prusiner, S. B., Bolton, D. C., Groth, D. F., Bowman, K. A., Cochran, S. P. & McKinley, M. P. (1982) Biochemistry 21, 6942-6950. 10. Prusiner, S. B., Groth, D. F., Bolton, D. C., Kent, S. B. & Hood, L. E. (1984) Cell 38, 127-134. 11. Oesch, B., Westaway, D., Walchli, M., McKinley, M. P., Kent, S. B. H., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B., Hood, L. E., Prusiner, S. B. & Weissmann, C. (1985) Cell 40, 735-746. 12. Chesebro, B., Race, R., Wehrly, K., Nishio, J., Bloom, M., Lechner, D., Bergstrom, S., Robbins, K., Mayer, L., Keith, J. M., Garon, C. & Haase, A. (1985) Nature (London) 315, 331-333. 13. Gabizon, R. & Prusiner, S. B. (1990) Biochem. J. 266, 1-14. 14. Riesner, D., Kellings, K., Meyer, N., Mirenda, C. & Prusiner, S. B. (1992) in Prion Diseases of Humans and Animals, eds.
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