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

Abstract

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that <25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.

Figure 1.

Discovery, design and characterization of ASOs used in this study. (A) 464 ASO candidates spanning the Prnp RNA sequence were screened in HEPA 1–6 cells, and Prnp RNA was quantified as previously described [28]. 305/464 (66%) of candidates screened in cells were ‘hits’ with a 95% confidence interval upper bound (based on N = 2 replicates) of <100% of untransfected controls. (B) The position, sequences and chemistries of previously reported active ASOs (1 and 2) (28), modified ASOs designed for the present study (5 and 6), and runner-up ASO candidates from design efforts undertaken for the present study (A, B and C). (C and D) Groups of N = 4 animals received a single 700 μg dose of the indicated treatment and ipsilateral cortex (C) or thoracic cord (D) mRNA was analyzed by qPCR 8 weeks later. (E) Body weight trajectories for animals shown in panels C and D, over the 8 weeks between dosing and tissue analysis. (F, G) Groups of N = 4 animals received a single 500 μg dose of the indicated treatment and ipsilateral cortex (F) or thoracic cord (G) mRNA was analyzed by qPCR 1 week later. (H) Groups of N = 4 animals received a 500 μg dose of the indicated ASO and ipsilateral cortex, thoracic cord, ipsilateral thalamus, or brainstem were analyzed by qPCR 1, 4 or 7 days later.

Figure 2.

Replication of early and late treatment efficacy of ASOs. Survival (A, D), body weights (B, E) and symptom trajectories (C, F) of mice treated with ASOs prophylactically (–14 and 76 dpi) (A–C) or at 120 dpi (D–F). Shaded areas represent 95% confidence intervals.

Figure 3.

Relationship between degree of PrP lowering and therapeutic benefit. (A) Dose versus ipsilateral cortical PrP mRNA knockdown determined by qPCR at 2 weeks post-treatment and normalized to the mean of saline-treated, non-infected animals, N = 3 per group, (B) PrP mRNA knockdown (from panel A) versus time to symptomatic endpoint in groups of N = 8 prion-infected animals receiving two injections of the indicated dose, at –14 and 76 dpi, and, for the same animals, (C) overall mortality, (D) body weights normalized to each mouse's individual weight at 122 dpi, (E) mean symptom count per animal and (F) mean nest score. Studies conducted at the Broad Institute. Shaded areas represent 95% confidence intervals.

Figure 4.

Natural history of RML prion infection. N = 12 mice infected with 30 μl of a 1% RML brain homogenate versus N = 12 uninoculated controls. In panels A–E, lines represent means, shaded areas represent 95% confidence intervals of the mean, and dots represent assessment timepoints. Nominal statistical significance thresholds (two-sided Kolmogorov-Smirnov test) are displayed as: * P < 0.05, ** P < 0.01, *** P < 0.001. (A) symptom accumulation (see Supplementary Figure S4 and Supplementary Table S3 for details), (B) nest-building scores, (C) weight change relative to each animal's 78 dpi baseline (see raw individual weights in Supplementary Figure S5A)†, (D) rotarod performance relative to each animal's –7 dpi baseline (see raw individual latencies in Supplementary Figure S5B), (E), plasma NfL (see raw individual NfL trajectories in Supplementary Figure S5C) and (F) overall mortality. †In panel C, prion-infected animals that reached endpoint between planned assessments and were weighed a final time prior to euthanasia are grouped together with animals at the next planned assessment timepoint — for example, animals that reached endpoint at 166 dpi are averaged into the 169 dpi timepoint. Studies conducted at McLaughlin Research Institute.

Figure 5.

Response of neuronal damage and astrocytosis biomarkers to ASO treatment at a pathological timepoint. (A) plasma NfL and (B) survival in wild-type mice infected with prions and dosed at 120 dpi, a timepoint at which the natural history study (Figure 4D) had indicated that NfL was dramatically elevated and rotarod performance and nest-building might be impaired. N = 10 per group, of which NfL was assessed in N = 10 saline-treated and N = 5 active ASO 6-treated animals. (C) live animal bioluminescence and (D) survival in Tg(Gfap-luc) mice infected with prions and dosed at 83–84 dpi, after two consecutive imaging sessions showed elevated luminescence in the RML group compared to uninfected controls. N = 9 per treatment group plus N = 14 uninfected controls. Shaded areas represent 95% confidence intervals. Studies conducted at McLaughlin Research Institute.

Figure 6.

Efficacy of PrP-lowering therapy is timepoint-dependent. Groups of N = 8 animals received saline or active ASO 6, chronically every ∼90 days beginning at the specified timepoint. Black triangle indicated when ASO was injected. (A–G) Survival time as a function of time of treatment initiation, (H) combined survival curves for saline-treated mice versus mice treated with active ASO 6 at early (–7 to 78 dpi) or late (105 to 120 dpi) timepoints. Survival curves for wild-type versus Prnp+/- animals infected with RML prions shown in Table 2 and Supplementary Figure S2 are reproduced here for comparison. Studies conducted at the Broad Institute. Shaded areas represent 95% confidence intervals.

Figure 7.

Effects of intervention at pathological and symptomatic timepoints. Animals were infected with RML prions and then received saline (N = 12) or a single 500 μg dose of active ASO 6 (N = 12) at the indicated timepoint. (A, E, I, M) survival; (B, F, J, N) individual body weight trajectories; (C, G, K, O) symptom count summarized by cohort; (D, H, L, P) nest-building activity summarized by cohort. For the 156 dpi timepoint, 7/23 animals (30%) reached endpoint prior to the intervention (red curve, panel M). Shaded areas represent 95% confidence intervals. Studies conducted at the Broad Institute.