Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry

Abstract

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have reproducibly shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. To date it has been unclear whether the reduced abundance of PrP in CSF results from its incorporation into plaques, retention in intracellular compartm ents, downregulation as a function of the disease process, or other factors. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to known concentrations of isotopically labeled standards. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In N=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N-and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. This confirms the findings from ELISA studies, demonstrating that lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

Multimodal small-molecule screening for human prion protein binders

“…The N-terminal methionine of HuPrP90-231 was mostly removed by endogenous proteases as expected based on the second residue being glycine 75 ; this was verified by intact protein LC-MS ( Figure S5C). HuPrP23-231, however, retains the N-terminal methionine 76 , again as expected given the second residue is lysine 75 . HuPrP90-231: MGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYP NQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGS S HuPrP23-231: MKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMH RYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQ RGSS…”

Antisense oligonucleotides extend survival of prion-infected mice

“…First, we find that efficacy against prion disease is achieved by 2 PrP-targeting ASOs, but not by a control ASO, demonstrating that ASO efficacy is due to lowering of PrP RNA and not due to aptameric interaction (35)(36)(37) between ASOs and PrP. This distinction is important because the PrP-lowering mechanism lends itself to measurement of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker (41,51). Second, we show a substantial survival benefit in mice treated with a single dose of a PrP-lowering ASO 120 dpi, when neuropathology is prominent and clinical signs imminent.…”

“…The benefit of ASOs in prophylactic treatment observed here may suggest a potential for PrP-lowering therapy to delay disease onset in presymptomatic individuals at risk for genetic prion disease. Predictive genetic testing makes it possible to identify individuals at >90% lifetime risk of prion disease (11), and while the variable age of onset poses a challenge for prevention trials (59), efforts are underway to enable informative clinical trials in this population (41,51,60). Meanwhile, the benefit of ASOs in late treatment may also suggest a potential for effective treatment in already-symptomatic individuals with prion disease.…”

Prion protein lowering is a disease-modifying therapy across prion disease stages, strains, and endpoints

“…This supports the universality of PrP lowering as a therapeutic strategy across strains and subtypes of prion disease. The above observations are also important because quantification of CSF PrP concentration 44,64,65 is being developed as a pharmacodynamic biomarker for PrP-lowering drugs. The validation of ASOs' mechanism of action in vivo, the tight relationship between degree of PrP lowering and disease delay, and the efficacy across prion strains, observed here all support the disease relevance of this biomarker 2 .…”