Antisense oligonucleotides extend survival of prion-infected mice

Abstract

Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrP; PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering one target mRNA in the brain, but their development for prion disease has been hindered by three unresolved questions from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here we test antisense oligonucleotides (ASOs) delivered by bolus intracerebroventricular injection to intracerebrally prion-infected wild-type mice. Prophylactic treatments given every 2-3 months extended survival times 61-98%, and a single injection at 120 days post-infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a non-targeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent, or even single, bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.

Keywords: Gene therapy; Neurodegeneration; Neuroscience; Prions; Therapeutics.

Figure 1. Potency and persistence of anti-PrP ASOs in the mouse brain.

(A) ASOs were administered i.c.v. at 500 or 700 μg to groups of n = 3 mice, and ipsilateral hemispheres were collected 8 weeks later and microdissected for regional PrP RNA quantification by RT-PCR (Supplemental Table 1). Data were normalized to the mean value for saline-treated animals. Error bars indicate 95%CI of the mean. (B) Western blots from whole contralateral hemispheres of SWR/R mice 8 weeks after treatment with 700 μg ASO. (C) RT-PCR quantification of PrP RNA in the ipsilateral cortex of n = 4 mice at 4, 12, and 16 weeks after a single 500 μg ASO dose. Data were normalized to the mean value for saline-treated animals. Error bars indicate 95%CI of the mean. (D) IHC images of sagittal midline cortical sections stained using antibodies to PrP (D13) or the ASO backbone at the indicated number of days after a single 300 μg i.c.v. injection of ASO. Representative images of a total of 3 mice per treatment group per time point, except saline 62–63 days, for which 5 mice were analyzed. Scale bar: 50 μm.

Figure 2. Benefit of prophylactic ASO treatment in prion-infected mice.

(A) Delay of onset of clinical signs characteristic of prion disease in animals treated at NIH. Arrows indicate timing of 300 μg i.c.v. doses. One ASO 1–treated mouse at NIH showed no clinical signs prior to euthanasia at 527 dpi (Figure 2C); its brain was negative for prion pathology by IHC and for prion seeding activity by RT-QuIC (75), suggesting that either the infection was cleared or the original inoculum had failed to deliver an infectious dose. (B) Disease duration (onset to end stage) in animals treated at NIH. Bars indicate mean and 95%CI of the mean. (C) All-cause mortality in animals treated at NIH. (D) Body weights of animals treated at the Broad Institute. Lines indicate means, and shaded areas indicate 95%CI of the means. Only time points with ≥ 2 animals are included. (E) All-cause mortality in animals treated at the Broad Institute. One control ASO-treated mouse at Broad Institute showed no symptoms prior to euthanasia at 315 dpi; its brain was positive by RT-QuIC, with endpoint at a 1 × 10^–7 dilution, suggesting it had eventually reached a prion titer similar to or slightly below that in terminal mice, but its survival > 15 SDs longer than the mean endpoint for its cohort suggests that the original inoculation had delivered an incomplete dose of prion infectivity. Arrows indicate timing of 500 μg i.c.v. doses. Treatment groups were n = 9 animals at NIH and n = 12 animals at Broad Institute, with some animals censored; see Supplemental Table 2 for details.

Figure 3. Benefit of ASO treatment against established prion infection in mice.

(A) Delay of clinical onset in animals treated at NIH. Arrow indicates timing of single 300 μg i.c.v. dose. (B) Disease duration (onset to end stage) in animals treated at NIH. Bars indicate mean and 95%CI of the mean. (C) All-cause mortality in animals treated at NIH. Arrow indicates timing of single 300 μg i.c.v. dose. Treatment groups were n = 9 animals.

Figure 4. Effects of PrP-lowering ASOs on prion neuropathology.

(A and B) Distributions of ASO, PrP, spongiosis (H&E), and astrogliosis (GFAP) in representative mice treated prophylactically (A) and at 120 dpi (B). Scale bar: 50 μm. Images are representative of a total of (A) 25 mice, including 7 saline, 4 control ASO, 5 each active ASO 1 and 2 terminal, and 2 each active ASO 1 and 2 comparator; and (B) 28 mice, including 3 no treatment, 9 saline, 7 control ASO, 7 active ASO 1 terminal, and 2 active ASO 1 comparator. Pixel counts from additional mice are provided in Supplemental Figures 3 and 5, and Western blots of PK-resistant PrP are provided in Supplemental Figures 4 and 6.