Prion Diseases

The development of in vitro seed amplification assays (SAA) detecting misfolded alpha-synuclein (αSyn) in cerebrospinal fluid (CSF) and other tissues has provided a pathology-specific biomarker for Lewy body disease (LBD). However, αSyn SAA diagnostic performance in early pathological stages or low Lewy body (LB) pathology load has only been assessed in small cohorts. Moreover, the relationship between SAA kinetic parameters, the number of αSyn brain seeds and the LB pathology burden assessed by immunohistochemistry has never been systematically investigated. We tested 269 antemortem CSF samples and 138 serially diluted brain homogenates from patients with and without neuropathological evidence of LBD in different stages by the αSyn Real-Time Quaking-Induced Conversion (RT-QuIC) SAA. Moreover, we looked for LB pathology by αSyn immunohistochemistry in a consecutive series of 604 Creutzfeldt-Jakob disease (CJD)-affected brains. αSyn CSF RT-QuIC showed 100% sensitivity in detecting LBD in limbic and neocortical stages. The assay sensitivity was significantly lower in patients in early stages (37.5% in Braak 1 and 2, 73.3% in Braak 3) or with focal pathology (50% in amygdala-predominant). The average number of CSF RT-QuIC positive replicates significantly correlated with LBD stage. Brain homogenate RT-QuIC showed higher sensitivity than immunohistochemistry for the detection of misfolded αSyn. In the latter, the kinetic parameter lag phase (time to reach the positive threshold) strongly correlated with the αSyn seed concentration in serial dilution experiments. Finally, incidental LBD prevalence was 8% in the CJD cohort. The present results indicate that (a) CSF RT-QuIC has high specificity and sufficient sensitivity to detect all patients with LB pathology at Braak stages > 3 and most of those at stage 3; (b) brain deposition of misfolded αSyn precedes the formation of LB and Lewy neurites; (c) αSyn SAA provides "quantitative" information regarding the LB pathology burden, with the lag phase and the number of positive replicates being the most promising variables to be used in the clinical setting.

Alzheimer's disease (AD) is characterized pathologically by amyloid-beta (Aβ) deposition in brain parenchyma and blood vessels (as cerebral amyloid angiopathy (CAA)) and by neurofibrillary tangles of hyperphosphorylated tau. Compelling genetic and biomarker evidence supports Aβ as the root cause of AD. We previously reported human transmission of Aβ pathology and CAA in relatively young adults who had died of iatrogenic Creutzfeldt-Jakob disease (iCJD) after childhood treatment with cadaver-derived pituitary growth hormone (c-hGH) contaminated with both CJD prions and Aβ seeds. This raised the possibility that c-hGH recipients who did not die from iCJD may eventually develop AD. Here we describe recipients who developed dementia and biomarker changes within the phenotypic spectrum of AD, suggesting that AD, like CJD, has environmentally acquired (iatrogenic) forms as well as late-onset sporadic and early-onset inherited forms. Although iatrogenic AD may be rare, and there is no suggestion that Aβ can be transmitted between individuals in activities of daily life, its recognition emphasizes the need to review measures to prevent accidental transmissions via other medical and surgical procedures. As propagating Aβ assemblies may exhibit structural diversity akin to conventional prions, it is possible that therapeutic strategies targeting disease-related assemblies may lead to selection of minor components and development of resistance.

Prion disease is a fatal neurodegenerative disease caused by the misfolding of prion protein (PrP) encoded by the PRNP gene. While there is currently no cure for the disease, depleting PrP in the brain is an established strategy to prevent or stall templated misfolding of PrP. Here we developed in vivo cytosine and adenine base strategies delivered by adeno-associated viruses to permanently modify the PRNP locus to achieve PrP knockdown in the mouse brain. Systemic injection of dual-adeno-associated virus PHP.eB encoding BE3.9max and single guide RNA installing PRNP R37X resulted in 37% average installation of the desired edit, 50% reduction of PrP in the mouse brain and 52% extension of lifespan in transgenic human PRNP mice inoculated with pathogenic human prion isolates representing the most common sporadic and genetic subtypes of prion disease. We further engineered base editing systems to achieve improved in vivo potency and reduced base editor expression in nontargeting tissues, resulting in 63% average PrP reduction in the mouse brain from a 6.7-fold lower viral dose, with no detected off-target editing of anticipated clinical significance observed in either human cells or mouse tissues. These findings support the potential of in vivo base editing as one-time treatment for prion disease.

Prion protein (PrP) lowering is effective in animal models of prion disease and is being tested clinically in prion disease patients, but there remains a need for more potent PrP-lowering drug candidates. Inspired by the reported potency and duration of action of divalent short interfering RNA (siRNA), a new oligonucleotide drug modality for the central nervous system, we sought to discover and develop a new PrP-lowering drug candidate. Herein we identify a mouse Prnp-targeting divalent siRNA molecule, 1682-s4, that lowers PrP to 49% residual brain expression in wild-type mice, and, in the context of intracerebral infection with Rocky Mountain Laboratories prions, achieves a 2.7-fold increase in survival time with pre-symptomatic chronic treatment and 64% increase in survival time with a single dose after symptom onset. We describe the generation of two transgenic mouse lines, Tg25109 and Tg26372, expressing the full human PRNP gene and its noncoding sequence, and demonstrate their utility for in vivo discovery of potent human PRNP-targeting oligonucleotides. We discover siRNA sequence 2439 against human PRNP and compare its potency in different divalent siRNA chemical scaffolds. We determine that both the fixed UU tail and extended nucleic acid linkages of scaffold s4 contribute to superior potency compared to other scaffolds tested, offering 9.4 and 15.9 percentage points respectively of additional PrP knockdown. A single dose of 348 µg of 2439-s4 lowered whole brain hemisphere human PrP in transgenic mice to 17% residual after 30 days, while 52 µg lowered PrP to 49% residual. A total of 1%-2% of the dose of 2439-s4 delivered into cerebrospinal fluid is retained in the brain, and the median effective tissue concentration is estimated at 1.2 μg per gram of tissue. Good Laboratory Practices toxicology studies identified no significant liabilities, and the US FDA has cleared an Investigational New Drug application to bring 2439-s4 into clinical trials.