20.109(S09) TR-yellow Mod3-proposal

Prions

Idea

• Do a time course analysis of human diseases, see if knockdown of expression of just one protein can be used to treat multiple prion diseases.
• Treat the whole brain (ie global treatment instead of local treatment)

Background

Prions are a mutated protein that binds to the endogenous form of the protein, forming plaques that aggregate in the brain and cause invariably fatal neurodegeneration. Prion disease can be contracted from exposure to the mutant protein through consumption or inadvertent transfusion of diseased samples.

All prion disease are different mutations in the same protein, so our hypothesis is that all can be treated via siRNA knockdown of the endogenous protein.

04/21/2009 Meeting minutes

Experimental steps:

1. pick prion disease (eg. mad cow)
2. knockdown expression of normal protein in (bovinized) mice, see if essential
3. expose to knockdown mice to prion pathogen

Our next steps:

1. research mad cow disease
   1. mechanism
   2. how it comes up as a disease
   3. to what it binds, etc

Papers to discuss next time

1. Kong, Q. RNAi: a novel strategy for the treatment of prion diseases. J Clin Invest. 2006 Dec;116(12):3101-3.
2. Pfiefer et al. Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice. J Clin Invest. 2006 Dec;116(12):3204-10
3. Mallucci et al. Targeting cellular prion protein reverses early cognitive deficits and neurophysiological dysfunction in prion-infected mice. Neuron. 2007 Feb 1;53(3):325-35.
4. Sutou et al. Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology. BMC Biotechnol. 2007 Jul 26;7:44.
5. Meslin et al. Silencing of prion protein sensitizes breast adriamycin-resistant carcinoma cells to TRAIL-mediated cell death. Cancer Res. 2007 Nov 15;67(22):10910-9.
6. White et al. Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease. Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10238-43. Epub 2008 Jul 16.

05/03/2009 Meeting Minutes

Possible experiments

• want to see if all human prion diseases (which are various mutations in PrnP) can be cured via knockdown of PrnP^C with siRNA
  • Creuztfeldt-Jakob Disease (CJD)
  • FFI (Fatal Familial Insomnia)
  • Kuru
  • Gertsmann-Straussler-Scheinker syndrome (GSS)

Discussion of papers

1. Basically a comment on Paper #2 in the same issue of the journal.
2. Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice.
   1. This paper was the first to successfully do lentivector-mediated RNAi treatment in prion-infected mice
   2. Summary: infected cells with scrapie, created chimeric mice. Treated those with siRNA that was injected into the striatum.
3. Point: targeting PrP^C mid-disease helps reverse cognitive deficits (no RNAi)
4. Point: bPRP can be knocked down (bovine cells)
5. Point: Silencing of PrP^C kills tumor cells (use in presentation when talking about implications since everyone loves cancer)
6. Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease.
   1. This paper pretty much paves the way for our work
   2. Summary: Treated prion-infected mice with lentiviral siRNA which was injected into the brain. Lentiviral treated mice lived longer than untreated ones.
   3. "Further exploration of the extent and timing of RNAi-mediated PrP knockdown required for increased therapeutic effect in prion disease can now be undertaken"

Experimental plan

1. Use siRNA against PrP in humanized mice infected with the four different prion diseases listed above. Compare the effectiveness of knocking down this protein in the survival of these mice.
2. Do a time course against Scrapie in sheep-ifed mice
   1. This is the further exploration suggested by paper #6

More papers of interest

1. Mallucci et al. Depleting Neuronal PrP in Prion Infection Prevents Disease and Reverses Spongiosis. Science. 2003 Oct 31;302(5646):871-4.
   1. Point: Depletion of PrP confers resistance to Prion disease.

05/10/2009 Meeting Minutes

• Need to finish making slides
  • Experimental procedures
    • siRNA against PRNP - does it work for the 4 diseases
    • Time Course analysis
  • Implications
    • Verify that this single siRNA can treat a variety of diseases
    • Elucidate optimal treatment regimen
  • Materials Needed
    • humanized mice with the PRNP gene that expresses PrP
    • lentiviral vector to inject the siRNA treatment