Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion

Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion

disease Melanie D. White*, Michael Farmer, Ilaria Mirabile, Sebastian Brandner, John Collinge, and Giovanna R. Mallucci† Medical Research Council (MRC) Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London, London WC1N 3BG, United Kingdom Edited by Charles Weissmann, The Scripps Research Institute, Jupiter, FL, and approved June 3, 2008 (received for review March 19, 2008)

Prion diseases are fatal neurodegenerative conditions for which there is no effective treatment. Prion propagation involves the conversion of cellular prion protein, PrPC, to its conformational isomer, PrPSc, which accumulates in disease. Here, we show effective therapeutic knockdown of PrPC expression using RNAi in mice with established prion disease.Asingle administration of lentivirus expressing a shRNA targeting PrP into each hippocampus of mice with established prion disease significantly prolonged survival time. Treated animals lived 19% and 24% longer than mice given an ‘‘empty’’ lentivirus, or not treated, respectively. Lentivirally mediated RNAi of PrP also prevented the onset of behavioral deficits associated with early prion disease, reduced spongiform degeneration, and protected against neuronal loss. In contrast, mice receiving empty virus or no treatment developed early cognitive impairment and showed severe spongiosis and neuronal loss. The focal use of RNAi therapeutically in prion disease further supports strategies depleting PrPC, which we previously established to be a valid target for prion-based treatments. This approach can now be used to define the temporal, quantitative, and regional requirements for PrP knockdown for effective treatment of prion disease and to explore mechanisms involved in predegenerative neuronal dysfunction and its rescue.

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Discussion The rescue of early neuronal dysfunction before neuronal loss is established is a clear goal for therapeutic intervention in neurodegenerative disease. Our previous findings that transgene-mediated PrP knockdown reversed predegenerative pathological changes and early behavioral deficits in prion disease led us to try to achieve the same effect therapeutically. PrPC knockout, both during development and postnatally, appears to be without detrimental effect (6, 21). We used RNAi to silence PrP expression in mice with established prion disease. Knockdown of PrP by RNAi (11) and resultant inhibition of PrPSc replication in cell culture have been described (12), and RNAi of PrP also works in vivo. Transgenic mice generated by lentiviral transduction of embryos stably express anti-PrP shRNAs and have increased resistance to prion infection because of RNAi-mediated reduced expression of endogenous PrP (13). However, until now, RNAi had not been used therapeutically in vivo in prion disease. Here, we have shown that treating mice with lentiviruses expressing shRNAs to knockdown PrP in established prion disease rescued early neuronal dysfunction and death in targeted areas and significantly prolonged survival. Injection of virus into the hippocampus 8 weeks after prion infection prevented the first behavioral deficits associated with early pathology of the CA1 region: loss of burrowing activity and object recognition memory (22) (Fig. 2). In our previous work, where PrP knockdown was due to recombination at the genomic level at 8 wpi, early deficits occurred but recovered rapidly in PrP-depleted animals. Here, injection of lentivirus expressing anti-PrP shRNAs at 8 wpi prevented their manifestation altogether, perhaps because posttranscriptional gene silencing is more rapid, or more tightly controlled temporally, than genetic excision of PrP encoding sequences after transgene expression. The benefits of RNAi treatment were also seen morphologically. There was significantly less spongiform degeneration and neuronal loss where anti-PrP lentivirus was delivered. These changes progress rapidly in RML-infected tg37 mice after 8 wpi, particularly in the hippocampus (4), and were marked in terminally ill LV-

Empty-treated animals at 12 wpi (Fig. 4). However, LV-MW1- treated mice culled up to 3 weeks later had minimal hippocampal spongiform change and neuronal loss (Fig. 4), suggesting sustained focal protection against neurotoxicity where PrP knockdown occurred. Interestingly, spongiosis was also reduced, although less significantly, in thalamus and cortex of animals treated with hippocampal injections (Fig. 5). PrPSc accumulation was also lower in animals with virally mediated RNAi of PrP in the hippocampus than in mock treated animals, and again this reduction was seen beyond the hippocampus, in thalamus and cortex. The more widespread changes are likely to reflect altered spread of prion infection after hippocampal PrP knockdown, as discussed below. Of note, PrPSc accumulation did not appear to affect neuronal function or survival, as reflected in preservation of hippocampal behaviors and structural neuronal integrity, and consistent with our observations in mice with Cre-mediated PrP depletion (5), which has implications for the level of knockdown required for therapeutic effect. Thus, simply slowing the rate of prion replication, here by reducing PrPC levels, may be effective for prevention of neurotoxic effects. The critical effect, however, was the effect of this treatment on survival of prion-infected mice. A single treatment with focal injection of virus resulted in significantly prolonged survival time of treated animals, compared with mock or untreated mice, with a mean increase in lifespan of 23.5% compared with untreated animals (Fig. 3). The spread of incubation times in the LV-MW1 group (87–129 days after inoculation, mean 105  4 days) is probably due to variation in neuronal transduction by virus seen in individual mice (data not shown) or variability between individual injections, with the highest levels of transduction affording the greatest protection and longest survival. The increased survival was strikingly large with respect to the very small volume of brain targeted. This may result from direct or indirect effects of localized neuroprotection or may simply be due to the reduction of PrP expression at a critical, or rate-limiting, site for prion replication. Prion incubation times are known to be inversely proportional to overall levels of PrP expression (3, 23, 24), and it is likely that regional variations also affect prion replication rates and incubation periods. The hippocampus is a focus of early prion replication and PrPSc deposition (see Fig. S3) both forRMLand other prion strains in various inbred lines and in some transgenic mice (25, 26), including tg37 mice, used here (4). We showed up to 80% reduction of hippocampal PrP mRNA expression (Fig. 1B) with single LV-MW1 administration; this localized knockdown may therefore eliminate a key area for early prion replication in this model. Further, we have found no evidence for the spread of lentivirus beyond the injection site, supporting the concept that it is the effect of localized hippocampal PrP depletion that alters the spread and replication of RML prions in this model. Clearly, all animals succumb eventually, presumably due to prion-mediated neurodegeneration in other critical brain regions, but the neuroprotective effects seen within the hippocampus and beyond are clearly a desirable effect of therapy. If transduction were to be more widespread, by pseudotyping lentiviruses with coat proteins that allow retrograde transport (27, 28) or using evolving mechanical techniques for enhanced delivery (29–31), more extensive neuroprotection and longer survival might ensue. Even focal targeting may have therapeutic application in some situations, however. In conclusion, we have used lentivirally mediated RNAi for treatment of established prion infection in mice. Even localized single administration of these viruses to the hippocampus prolonged the lifespan of infected mice, protected transduced neurons from degenerating, reduced PrPSc accumulation, and prevented the onset of the first behavioral deficits associated with the disease. Our findings urther support therapeutic strategies directed at PrP knockdown for the treatment of prion diseases and are also relevant for neurodegeneration more widely, highlighting the importance of intervention when neuronal dysfunction can still be reversed. The approach used here paves the way not only for possible future therapy but also for mechanistic dissection of toxicity and recovery in prion diseases. Further exploration of the extent and timing of RNAi-mediated PrP knockdown required for increased therapeutic effect in prion disease can now be undertaken. Methods...snip...end

White et al. PNAS
July 22, 2008
vol. 105
no. 29
10243 NEUROSCIENCE

www.pnas.orgcgidoi10.1073pnas.0802759105

Author contributions: G.R.M. designed research; M.D.W., M.F., I.M., and S.B. performed research; M.D.W., M.F., I.M., S.B., J.C., and G.R.M. analyzed data; and G.R.M. wrote the paper. Conflict of interest statement: J.C. is a director and shareholder of D-Gen Limited, an academic spin-out company working in the field of prion disease diagnosis, decontamination, and therapeutics. D-Gen markets the ICSM35 and ICS18 antibodies used in this study. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. *Present address: Centre for Neuroscience Research, University of Edinburgh, EH8 9XD, United Kingdom. †To whom correspondence should be addressed. E-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000004/!x-usc:mailto:g.mallucci@prion.ucl.ac.uk. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0802759105/DCSupplemental. © 2008 by The National Academy of Sciences of the USA



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