Study design and sites. We sought to determine whether ASOs could sequence-specifically lower PrP in the mouse brain and extend survival in prion-infected mice. Scientists at Ionis Pharmaceuticals led the discovery of ASOs and characterization of their potency by reverse transcription PCR (RT-PCR) in both cells and animals. Experiments in prion-infected animals were conducted concurrently at 2 sites — NIH/NIAID/Rocky Mountain Laboratories and the Broad Institute of MIT and Harvard. NIH also characterized ASO potency effects on PrP by protein quantification and IHC.

ASO treatment studies were designed as controlled laboratory studies, with a primary endpoint of terminal prion disease requiring euthanasia. At NIH, animals were also monitored for clinical onset according to prespecified clinical neurological signs: progressive deterioration of ataxia, tremors, myoclonus, weight loss, somnolence, kyphosis, and poor grooming. At NIH, some animals were prespecified for intercurrent euthanasia to serve as time point–matched controls in biochemical and histological analyses, and they were excluded from analyses of prion disease onset and survival. Animals that died of nonprion causes were included in calculations of all-cause mortality but were excluded from analyses of prion disease symptom onset. At Broad Institute, animals were monitored once per week, increasing to every other day after 120 dpi, for the following signs: generalized tremor, ataxia, difficulty righting from a supine position, rigidity of the tail, stare or blank look, and hindlimb weakness; animals were weighed every other day after 120 dpi and were euthanized upon body condition score <2, body weight loss >20%, inability to reach food or water, severe respiratory distress, or severe neurological deficits.

Animals. Experiments used 6- to 12-week-old WT female mice, either C57BL/6N purchased from Taconic Biosciences Inc. or SWR/R mice, an outbred colony of primarily Swiss origins maintained at Rocky Mountain Laboratories for many generations (69). Both mouse strains harbor the Prnpa (MoPrP-A) haplotype (70), as found in the mouse reference genome. Figure 1, A, B, and D; Figure 2, A–C; Figure 3; Figure 4; and Supplemental Figures 2–6 display data from SWR/R mice. Figure 1, C–E, and Supplemental Figure 1 display data from C57BL/6N mice.

ASO synthesis, screening, and lead identification. Synthesis and purification of all chemically purified ASOs were performed as previously described (71). Approximately 500 ASOs were designed against the full mouse Prnpa gene. Electroporation of ASOs was carried out using the HT-200 BTX Electroporator with ElectroSquare Porator (ECM830) voltage source at 135 V in 96-well electroporation plates (BTX, 2 mm; Harvard Apparatus). ASOs were screened in HEPA1-6 cells (ATCC, 1830) at 7 μM. Cells were harvested at 24 hours after treatment for RNA extraction, and mouse Prnp mRNA was quantified by RT-PCR. The most potent ASOs were taken into a 4-point dose response in HEPA1-6 cells. Active ASO 1 and active ASO 2 were then characterized by screening in C57BL/6N mice by i.c. ventricular injection of 300 μg, with tissue collection at 2 weeks after dose for Prnp mRNA reduction (Supplemental Figure 1A). After synthesis, ASOs were aseptically diluted to 100 mg/ml in PBS and frozen at –20°C.

Stereotactic i.c.v. injection of ASO or PBS at the NIH. After thawing, the actual concentrations of each ASO were determined by spectroscopy at an absorbance of 260 nm. Based on absorbance calculated concentration values, each ASO was further diluted to 66.7 μg/μl in PBS, and 4.5 μl was injected i.c.v. into each mouse as described below. For buffer-only control mice, 4.5 μl of PBS was injected i.c.v. Aliquots of the ASO solutions were stored at –20°C for repeated ASO treatments, and a fresh aliquot was thawed and used for injections at each of the designated time points.

Two- to 3-month-old female mice were anesthetized with isoflurane and prepared for surgery by shaving the hair from the dorsal surface of the skull and applying chlorhexidine-based surgical scrub (BD Biosciences) to the area. Mice were then positioned on a stereotaxic frame (David-Kopf Instruments) and maintained on isoflurane anesthesia. Using aseptic technique, a 1-cm midline incision was made in the skin over the dorsal surface of the skull, and the skull was exposed to allow positioning of the drill over the bregma point of reference. Coordinates used from bregma were 0.0 mm anterioposterior, 0.8 mm lateral (right), and 2.5 mm ventral (down) to skull surface. These coordinates were selected to target the center of the lateral ventricle. A small hole was drilled in the surface of the skull prior to placement of the 32-gauge delivery needle (World Precision Instruments). A total volume of 4.5 μl containing 300 μg of ASO in PBS or PBS alone was injected into the ventricle at a rate of 1 μl/sec using an UltraMicroPump III with Micro4 pump controller (World Precision Instruments). The needle was kept in place for 1 minute following injection to minimize reflux. The skin incision was closed with suture. Mice were recovered in heated cages after surgery and received a single s.c. injection of 0.2 mg/kg buprenorphine (Par Pharmaceuticals) for postoperative pain management. Patency of the needles was verified prior to and after injections.

Stereotactic i.c.v. injection of ASO or PBS at Ionis Pharmaceuticals and the Broad Institute. Animals were induced with 3% isoflurane and maintained on 3% isoflurane for a surgical plane of anesthesia throughout the procedure. Mouse heads were shaved and swabbed with betadine, and animals received prophylactic meloxicam for pain relief. Animals were placed in a stereotaxic apparatus (ASI Instruments, SAS-4100) with the 18° ear bars in the ear canals and the incisors in the tooth bar of the mouse adapter, adjusted to –8 mm so that the bregma and lambda landmarks on the skull were level. After making an approximately 1-cm incision in the scalp, s.c. tissue and periosteum were scrubbed from the skull with sterile cotton-tipped applicators to reveal the bregma. Hamilton syringes (VWR 60376-172) fitted with 22-gauge Huber point removable needles (VWR 82010-236) were filled with 10 μl of saline with or without ASO (diluted from 100 mg/ml in DPBS, Thermo Fisher Scientific, 14190). The needle was positioned over bregma and then moved to coordinates 0.3 mm anterior, 1.0 mm right, and 3.0 mm down after the bevel of the needle disappears through the skull. Saline (10 μl) was injected gradually by hand over approximately 10 seconds. After 3 minutes, the needle was backed out of the skull while applying downward pressure on the animal’s skull with a sterile cotton-tipped applicator. The incision was sutured with 1 horizontal mattress stitch using 5-O Ethilon suture (Ethicon, 661H). Animals were allowed to recover from the anesthesia in their home cage. For the data in Figure 2, D–E, the first round of injections were performed at Ionis Pharmaceuticals at –14 dpi, and animals were then shipped to the Broad Institute for prion inoculation and a subsequent second round of i.c.v. injections at 76 dpi (90 days after the first injections).

I.c. prion inoculations at the NIH. Per the time course shown in Figures 2 and 3, 8 to 12-week-old female mice were injected i.c. with 25 μl of 1% brain homogenate in PBS prepared aseptically from a pool of 10 RML prion–infected mouse brains excised at terminal stage of rodent-adapted scrapie (1 × 108.3 ID 50 /gram of brain). A 10% brain homogenate was aseptically prepared in 0.32 M sucrose by douncing the pool of excised brains 10 times each, first with the loose pestle and then with the tight pestle (Wheaton glass); sonicated for 2 pulses of 1 minute each (held for 30 seconds on ice in between pulses) in a cuphorn sonicator at maximum setting, followed by centrifugation at 1,500 g for 5 minutes; and resulting supernatant was used for inoculations. Inocula was aliquoted and stored at –80°C, and a fresh aliquot was used for each set of inoculations after it was rapidly thawed at 37°C, sonicated in a cuphorn sonicator at maximum setting for 2 sonication pulses as before, and dilution was done in PBS (Amresco). This was made and held at room temperature just prior to inoculations. Mice were restrained during i.c. injections by anesthesia using saturated isofluorane vapors in a bell jar until the mice were unconscious.

I.c. prion inoculations at the Broad Institute. Animals were 7–10 weeks old at the time of inoculation so that skulls were cartilaginous enough to allow manual i.c. inoculation. Each animal received 30 μl of a 1% RML prion brain homogenate, extruded through successively smaller-gauge needles, and irradiated at approximately 7.0 kGy to kill opportunistic pathogens prior to injection (72). Brain homogenate was loaded into a 300-μl BD SafetyGlide Insulin 31G syringe with a 6 mm needle (BD Biosciences, 328449). Mice were induced and maintained on a surgical plane of anesthesia with 3% isoflurane. Mouse heads were wiped with betadine. The needle was manually inserted through the skull, the plunger was depressed, and, after 3 seconds, it was removed. Animals were allowed to recover in their home cages.

RT-PCR. Cultured cells were lysed in 300 μl of RLT buffer (Qiagen) containing 1% (v/v) 2-mercaptoethanol (BME, MilliporeSigma). “For RNA extraction, the following were dissected: (a) a 2-mm coronal section of the cortex at 1 mm posterior to the injection site, (b) the hippocampus, and (c) a 2-mm coronal section of the thoracic spinal cord. Tissues were homogenized in 500 μl of RLT buffer containing 1% (v/v) BME. RNA was isolated from 20 μl of lysate with an RNeasy 96 Kit (Qiagen) that included in-column DNA digestion with 50 U of DNase I (Invitrogen). RT-PCR was performed using StepOne Realtime PCR system (Applied Biosystems), as described previously (50). The sequences of primers and probes are provided in Supplemental Table 1. PCR results were normalized by housekeeping gene cyclophilinA/Ppia and further normalized to the level in PBS-treated mice or untreated cells.

PrP immunoblot analysis of brain tissues for total PrP and PK-resistant PrP. After euthanasia and dissection, half of a sagittally divided mouse brain was flash frozen in liquid nitrogen and stored at –80°C; the other half was put into neutralized 10% formalin and used for immunohistochemical analysis. For immunoblot analysis, brains were thawed on ice. While thawing, the mass of each brain sample was determined; then, cold sterile phosphate buffered saline was added in order to make a 20% (weight/volume [w/v]) brain homogenate in a 2-ml polypropylene microcentrifuge tube containing 0.6 mg of 1 mm zircon beads (BioSpec). The tubes were then shaken in a bead beater (Bead Mill 24, Thermo Fisher Scientific) at maximum setting for 1 minute and placed on ice. Immediately afterward, the samples were centrifuged for 2 minutes at 2,000 g. The supernatant was aliquoted and returned to ice if immunobloted immediately or frozen at –80°C. To determine amount of total PrP, the brain homogenates were diluted to 1% in 0.04 ml total volume in 1× Sabu (0.0625 M Tris-Cl [pH 6.8], 0.003 M EDTA, 5% glycerol, 5% SDS, 4% β-mercaptoethanol, 0.02% bromophenol blue [MilliporeSigma]), vortexed well, and boiled for 5 minutes. Of the resulting sample, 10 μl was loaded onto 10% Bis-Tris NuPAGE polyacrylamide gels (Invitrogen) for electrophoresis. For analysis of PK-resistant PrP, 5 μl of 20% brain homogenate was diluted into 0.1 M Tris-Cl (pH 8.5), 0.15 M NaCl, 0.001 M CaCl 2 , and 50 μg/ml PK (Calbiochem) and incubated at 37°C for 1 hour. Then, 1 μl of 0.1 M Pefabloc (Fluka) was added; samples were vortexed and incubated on ice for 5 minutes; 55 μl of 2× Sabu was added, vortexed, and boiled for 5 minutes; and 10 μl of each sample was loaded onto gels as above. All gels were blotted onto PVDF membranes (MilliporeSigma) in Towbin buffer (0.025 M Tris base, 0.192 M glycine, 20% methanol) using a semidry blotter (Biometra) per manufacturer’s recommendations. Membranes were placed into a 50-ml polypropylene conical tube with the protein side facing inward and blocked using 5% Blotting-Grade Blocker (Bio-Rad) in TBST buffer (0.01 M Tris base, 0.15 M NaCl, 0.05% Tween 20) for 1 hour with continuous gentle rolling rotation. Blocking solution was removed and replaced with 10 ml mAb 6D11 (Santa Cruz Biotechnology Inc.) diluted 1:5,000 in blocking solution and incubated for 1 hour at room temperature. The membranes were washed in the same tube for 5 minutes, 4 times sequentially using 40 ml TBST dispensed into the tube. Next, the membranes were incubated/rotated for 1 hour in 10 ml goat anti–mouse alkaline phosphatase (Thermo Fisher Scientific, A16069) diluted 1:10,000 in blocking solution. After this incubation, membranes were washed 5 times for 5 minutes sequentially as above. Membranes were placed protein-side down into a square petri dish with 1.25 ml of AttoPhos solution (Promega) for 5 minutes and then air-dried overnight while hanging from a clip. Membranes were placed into a nonfluorescing plastic sheet protector and scanned with a Typhoon FLA 9500 (GE Healthcare) fluorescence imager. Resulting images were quantified using ImageQuant software (GE Healthcare).

IHC and histology. Brains were removed and cut in half in the sagittal plane, and one half of each brain was placed in 10% neutral buffered formalin for 3–5 days. Tissues were then processed by dehydration and embedding in paraffin. Sections were cut using a standard Leica microtome, placed on positively charged glass slides, and air-dried overnight at room temperature. On the following day, slides were heated in an oven at 60°C for 20–30 minutes.

For PrP, glial fibrillary acidic protein (GFAP), and ASO immunohistochemical staining, all deparaffinization, antigen retrieval, and staining were performed using the Ventana automated Discovery XT stainer. PrPSc staining requires aggressive antigen retrieval using high temperatures to expose the epitopes. Antigen retrieval for PrPSc staining was performed by incubation in CC1 buffer (Ventana) containing Tris-borate-EDTA (pH 8.0; MilliporeSigma), for 100 minutes at 95°C. Immunohistochemical staining for PrP was done using human anti–PrP monoclonal D13 antibody (73) in tissue culture fluid at a dilution of 1:100 for 2 hours at 37°C. The secondary antibody was biotinylated goat anti–human IgG at a 1:250 dilution (Jackson ImmunoResearch), and streptavidin-biotin peroxidase was used with DAB as chromogen (DAB Map kit; Ventana Medical Systems). For GFAP staining of astrocytes, antigen retrieval was done by using the Discovery XT system with the mild CC1 protocol (cell conditioning buffer containing Tris-borate-EDTA [pH 8.0], with incubation for 12 minutes at 100°C). The anti-GFAP antibody (74) was used at a dilution of 1:3,500 in antibody dilution buffer (Ventana, ADB250), applied for 16 minutes at 37°C. The secondary antibody was biotinylated goat anti–rabbit IgG (Biogenex Ready-to-use Super Sensitive Rabbit Link, HK3369R), applied for 16 minutes at 37°C. Staining was completed by using a RedMap detection kit. To stain ASO distribution in tissues, slides were pretreated with PK (DAKO Ready-to-Use) for 4 minutes at 37°C. The previously described (22) anti-ASO antibody (New Zealand rabbit polyclonal serum #6651, Ionis Pharmaceuticals) was applied at a 1:20,000 dilution in antibody dilution buffer (Ventana ADB250) for 60 minutes at 37°C. The secondary antibody was biotinylated goat anti–rabbit IgG (Biogenex, HK3369R) applied for 32 minutes at 37°C. The DAB Map Kit was applied as described above. For all IHC slides, hematoxylin was used as the counterstain and antibody diluent alone was used as a negative control. H&E staining was performed according to the manufacturer’s (Shandon) instructions: hematoxylin incubation of 12 minutes and eosin incubation of 4 minutes. Sections stained with D13, anti-GFAP, anti-ASO, and H&E were scanned with an Aperio ScanScope XT (Aperio Technologies Inc.), and they were analyzed and photographed using Aperio Imagescope software. Sections that were IHC stained for ASO and PrP were scanned with an Aperio ScanScope XT (Aperio Technologies Inc.) and quantified using the ImageScope positive pixel count algorithm (version 9.1). For each brain, a 5-μm–thick median sagittal section representing approximately 55 mm2 was evaluated. The pixel-counting algorithm interpreted the darkness or lightness of each pixel intensity and divided the data into categories based on intensity. The darkest staining possible (black) was given a score of 0, and the lightest staining (white) pixel score was 255. Hues of brown produced by DAB IHC detection systems used for the ASO and PrP staining score positive using this algorithm. For both ASO and PrP pixel scoring, all positive pixels (scores of 0–220) and negative pixels (scores of 221–255) were counted. To calculate the percentage of positive pixels in each tissue section, the following formula was used: positive pixels/total pixels × 100.

Statistics. For survival experiments, disease onset and survival endpoints were plotted as Kaplan-Meier survival curves. Differences between groups were considered visually obvious; statistical tests were not used. For ASO-mediated RNA reduction quantification, days from onset to terminal disease, and quantification of immunohistochemical markers, 95% CIs were calculated as ±1.96 times the SEM. Data were analyzed using GraphPad Prism (NIH) or R 3.5.1 (Broad Institute). Data and source codes sufficient to reproduce the figures herein are provided in a GitHub repository (https://github.com/ericminikel/aso_survival, under release 1.0; https://github.com/ericminikel/aso_survival/releases/tag/v1.0).

Study approval. All experimental procedures involving animals were approved by IACUCs (Ionis IACUC protocol P-0273, Broad Institute IACUC protocol 0162-05-17, and NIH IACUC protocol 2015-061) and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011).