Cell culture
Human B-lymphocytes and fibroblasts derived from the same homozygous patient with Huntington’s disease (HD) (GM04856/GM04857) and a healthy donor (GM07492/GM07491) (Coriell Institute for Medical Research), human neuroblastoma (SH-SY5Y) cells (ATCC®), human embryonic kidney 293 (HEK293) cells (ATCC); Madin–Darby Canine Kidney (MDCK) cells (ATCC®); MDCK cells expressing multidrug-resistance mutation 1 (MDCK-MDR1) (Absorption Systems); mouse CT26 cells (ATCC) were all grown at 37 °C in a humidified 5% CO2 atmosphere. Fibroblasts were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% antibiotic cocktail (penicillin-streptomycin/Thermo Fisher Scientific). All cell lines tested negative for mycoplasma contamination. Following the purchase of the cell lines, none of the cell lines was further authenticated. No commonly misidentified cell lines were used in the study.
High-throughput screening for the identification of HTT-lowering molecules
Human fibroblasts derived from a homozygous patient with HD (GM4857) were grown for 96 h in the presence of test compounds (in 0.5% DMSO) or controls at 37 °C in 5% CO2. After 96 h, cells were lysed and frozen. Huntingtin protein (HTT) levels were measured in lysates as described below. Compounds that decreased HTT levels relative to DMSO control were further tested in full dose–response.
Quantification of HTT protein in cells
For analysis in the ECL assays, test compounds were serially diluted with a threefold step in 100% DMSO (Sigma®) to generate a seven-point concentration–response curve. A solution of test compound (500 nL, 200× in DMSO) was added to each test well; the final concentration of DMSO was 0.5%. Fibroblasts were seeded in 96-well flat-bottomed plates (Thermo Fisher Scientific) at 4 × 103 cells/well in 100 µl of culture medium containing the test compound or DMSO vehicle control and incubated for 96 h (37 °C, 5% CO2, 100% relative humidity). After removal of the supernatant, cells were lysed in 50 μL of 1× Lysis Buffer11 (LB11) extraction buffer (50 mM Tris [pH 7.4], 300 mM NaCl, 10% [w/v] glycerol, 3 mM ethylenediaminetetraacetic acid (EDTA), 1 mM MgCl2, 20 mM glycerophosphate, 25 mM NaF, 1% Triton X-100), containing a Complete™ protease inhibitor cocktail (Roche Diagnostics®) with shaking at 4 °C for 30 min; the plates were then stored at −20 °C.
For western blot analysis, fibroblasts were plated at 5 × 104 cells/well in 1 mL 10% FBS/DMEM with GlutaMAX™ supplement (Thermo Fisher Scientific) in 24-well plates (Thermo Fisher Scientific) and incubated for 3–4 h (37 °C, 5% CO2, 100% relative humidity). Cells were then treated with HTT-C1 at different concentrations (0.5% DMSO) in triplicate wells for 96 h. Cells were lysed in 75 μL Laemmli buffer (Bio-Rad Laboratories, Inc.).
ECL protein assay
MESO SCALE DISCOVERY® 96-well plates (MSD®) were coated overnight at 4 °C with primary antibodies in phosphate-buffered saline (PBS; 30 µl/well). The plates were washed three times with 0.05% Tween-20 in 1× PBS (PBS-T; 200 µl/well) then blocked (100 µl/well; 5% bovine serum albumin (BSA) in PBS-T) for 5–6 h at room temperature with shaking. Plates were then washed three times with PBS-T. Cell lysates were transferred to the antibody-coated plates (25 µl/well) and incubated with shaking overnight at 4 °C. After removal of the lysates, the plates were washed three times with PBS-T, and 25 µl of detection antibody in 1% BSA, PBS-T was added to each well and incubated with shaking for several hours at room temperature. After three washes with PBS-T, 25 µl of SULFO-TAG secondary antibody (MSD; 0.25 µg/ml in 1% BSA, PBS-T) was added to each well and incubated with shaking for 1 h at room temperature. After washing three times with PBS-T, 150 µl of read buffer T with surfactant (MSD) was added to each empty well and the plate was imaged on the SI 6000 imager (MSD) according to manufacturers’ instructions for 96-well plates. Primary capture antibodies included: anti-polyglutamine-expanded HTT mouse monoclonal antibody (mAb) at 1 µg/mL (clone MW1; Developmental Studies Hybridoma Bank); anti-HTT mAb at 1 µg/mL (clone 1HU-4C8; Millipore; catalogue # MAB2166); anti- Kirsten rat sarcoma viral oncogene homologue (KRAS) rabbit polyclonal antibody at 1 µg/ml (Abcam; catalogue # ab137739). Detection antibodies included: Huntingtin (D7F7) XP® Rabbit mAb at 0.25 µg/ml (Cell Signalling Technology®; catalogue # 5656); anti-human-KRAS mAb at 0.25 µg/ml (clone 2C1; LSBio; catalogue # LS-C175665-100).
RT-qPCR quantification of HTT mRNA in cells
Test compounds were serially diluted threefold in 100% DMSO to generate a seven-point concentration curve. A solution of test compound (500 nL, 200× in DMSO) was added to each test well. Fibroblasts were seeded in 96-well flat-bottomed plates (Thermo Fisher Scientific) at 1 × 104 cells/well in 100 µl of culture medium containing the test compound or DMSO vehicle control and incubated for 24 h (37 °C, 5% CO2, 100% relative humidity). After removal of the supernatant, cells were lysed in RNA lysis buffer (1 M Tris-HCL pH 7.4, 5 M NaCl, 10% IGEPAL®CA-630; 50 µL/well) for 1 min at room temperature, before 50 µL of chilled nuclease-free water was added to each well; plates were then transferred immediately onto the ice before storing at −80 °C overnight.
The mRNA levels of the huntingtin gene (HTT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified using TaqMan-based RT-qPCR primers and probes (Thermo Fisher Scientific; Supplementary Table 1) and the AgPath-ID™ one-step RT-PCR Kit (Thermo Fisher Scientific). RNA samples were transferred (2 μL/well) to the Armadillo 384-well PCR plate (Thermo Fisher Scientific) containing 8 μL/well of the AgPath-ID™ one-step RT-PCR reaction mixture (Thermo Fisher Scientific) in a final volume of 20 μL. The plate was then sealed with MicroAmp™ Optical Adhesive Film (Thermo Fisher Scientific) and placed in the CFX384 Touch™ Real-Time PCR thermocycler (Bio-Rad Laboratories, Inc.). RT-qPCR was carried out at the following temperatures for indicated times: Step 1: 48 °C (30 min); Step 2: 95 °C (10 min); Step 3: 95 °C (15 s); Step 4: 60 °C (1 min); then Steps 3 and 4 were repeated for a total of 40 cycles.
Western blot analysis
HD fibroblasts (GM04857) were treated with compounds and lysed in 70 μL sample buffer (24-well plates/Thermo Fisher Scientific). Medium was aspirated, rinsed once with PBS, 70 μL Invitrogen™ NuPAGE™ Sample Buffer was added, rocked at room temperature for 10 min, and lysates frozen. Cell lysates were boiled for 10 min—loaded 45 μL/well. Cell lysates (45 µl/lane) were separated using pre-cast 3–8% Tris-Acetate gels (NuPAGE™ 3–8%, Tris-Acetate, 1.0 mm, Midi Protein Gel, 12 + 2-well; Thermo Fisher Scientific) for 5–6 h at 130 V. After electrophoresis, proteins were transferred to nitrocellulose membranes (0.45 μM nitrocellulose/Bio-Rad) at 150 mAp in NuPAGE Transfer Buffer (20× diluted to 1×; Thermo Fisher Scientific) for 90 min at 4 °C. Membranes were blocked overnight in blocking buffer (Li-Cor blocking buffer + 0.1% Tween) at 4 °C, washed four times (for 5 min) in PBS + 0.1% Tween (PBS-T; Thermo Fisher Scientific), and incubated overnight at 4 °C with primary antibodies (in Li-Cor blocking buffer + 0.1% Tween). Blots were washed with PBS-T, probed with secondary antibodies (in Li-Cor blocking buffer + 0.1% Tween) at room temperature for 1 h and washed again with PBS-T. Bound antibodies were visualised using the Odyssey® imaging system (LI-COR) according to the manufacturer’s instructions. The following primary antibodies were used: anti-HTT at 1:1000 (clone 1HU-4C8; Millipore; catalogue # MAB2166), anti-utrophin (UTRN) at 1:250 (clone DRP3/20C5; Vector Laboratories; catalogue # VP-U579), anti-oxidoreductase-protein disulphide isomerase (PDI) at 1:10,000 (Santa Cruz; catalogue # SC20132), anti-βactin at 1:10,000 (clone AC-74; Sigma; catalogue # A2228), anti-GAPDH at 1:1000 (Thermo Fisher Scientific; catalogue # PA1-987), anti-alpha serine/threonine-protein kinase (AKT) at 1:1000 (Cell Signalling; catalogue # 9272). Secondary antibodies included: Alexa Fluor® 680 goat anti-mouse immunoglobulin G (IgG) at 1:10,000 (Thermo Fisher Scientific; catalogue # A28183), IRDye® 800CW donkey anti-mouse IgG at 1:10,000 (Li-Cor; catalogue # 926-32212), and IRDye® 800CW donkey anti-rabbit IgG at 1:10,000 (Li-Cor; catalogue # 925-32213).
Primer walking assay, endpoint RT-PCR and AmpliSeq analysis
B-lymphocytes (GM04856 cells) were plated in six-well plates (Thermofisher) at 5 × 105 cells/well in 2 mL of 10% FBS, DMEM and incubated for 6 h (37 °C, 5% CO2, 100% relative humidity). Cells were then treated with HTT-C1 at 125 nM (in 0.5% DMSO) in triplicate for 24 h. RNA was purified with the RNeasy Mini Kit (Qiagen) using the manufacturer’s protocol. Samples were prepared for RT-PCR (described previously in Methods) using 0.04 µL of each primer (at 100 µM). For reverse transcription and PCR, the following steps were performed: reverse transcription step: 48 °C (15 min); PCR steps: Step 1: 95 °C (10 min), Step 2: 95 °C (30 s), Step 3: 55 °C (30 s), Step 4: 68 °C (1 min); Steps 2–4 were repeated for 34 cycles, then held at 4 °C. PCR products were separated on 2% pre-cast agarose E-gels (Invitrogen), stained with ethidium bromide and visualised using a UVP gel imager (Thermo Fisher Scientific). Primer sets used for primer walking are provided in Supplementary Table 2.
Ion AmpliSeq analysis of HTT pre-mRNA splicing GM04856 cells were plated in six-well plates at 5 × 105 cells/well in 2 mL of 10% FBS, DMEM and incubated for 6 h (37 °C, 5% CO2, 100% relative humidity) before treatment with 125 nM HTT-C1 (in 0.5% DMSO) in triplicate for 24 h. Cellular RNA was then extracted and purified (described previously in Methods). The Ion AmpliSeq technology (Thermo Fisher Scientific), which is a PCR-based target enrichment and next-generation sequencing platform, was used as a targeted measure of exons across the entire HTT transcript, with the goal of monitoring the presence of any (and all) novel splice variants in the presence of compound. PCR enrichment of HTT exon targets was accomplished by applying a custom HTT AmpliSeq panel (PTC proprietary primer set). The panel consisted of two separate PCR primer pools, each producing 33 amplicons. The complete HTT assay has 66 amplicons (mean size, 135 bp) covering all 67 exons. The AmpliSeq workflow (Supplementary Fig. 11) included: (a) RNA reverse transcription, (b) target amplification, (c) partial primer digestion, (d) adapter ligation, (e) library amplification, (f) sequencing reaction, (g) sequencing data analysis. For data analysis, AmpliSeq reads (Fastq format) were mapped to human genome (hg19) using TopHat232 which allows identification of both known and novel splice junctions. For each one of the 66 introns of the HTT gene, we calculated a JEI (Fig. 2c) using the percent of read supporting the splicing of the exact annotated intron among all reads supporting the splicing isoforms using either the 5′ss or/and 3′ss of that intron. A JEI value of 100% indicates full splicing of the intron. A JEI value <100% indicates alternative splicing paths (eg. inclusion of a cryptic exon or use of alternative 5′ or 3′ss). Biological triplicates were performed for each treatment group. We compared the difference of JEIs between compound and DMSO treated samples using the Student’s t test.
Splice-site score
The 5′ and 3′ss MAXENT scores were calculated using MaxEntScan33 (http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html) representing the strength of splice sites.
Analysis of nonsense-mediated decay
GM04856 cells were treated with DMSO or 250 nM HTT-C1. After 18 h, cells were treated with 10 µM cycloheximide (Sigma) or DMSO. Total RNA was isolated after 2, 4 and 8 h and analysed by endpoint PCR (described previously in Methods).
Transfection
Wild-type and mHTT and U1 minigene constructs were designed at PTC and synthesised at GenScript®. For U1 constructs 5 × 105 HEK293 cells were transfected with 2 µg of plasmid deoxyribonucleic acid (DNA) or mock control in six-well plates, using 6 µl Fugene6® (Promega) according to the manufacturer’s instructions; after incubating for 24 h (37 °C, 5% CO2, 100% relative humidity), cells were treated with either 1 µM HTT-C1 or 0.5% DMSO control and incubated for 48 h. For HTT constructs, 5 × 105 HEK293 cells were transfected with 50 ng of plasmid DNA in 24-well plates, using 6 µl Fugene6® according to the manufacturer’s instructions. After incubating overnight (37 °C, 5% CO2, 100% relative humidity), cells were treated with varying concentrations of compounds in a final concentration 0.05% DMSO and incubated for 24 h.
RNA-Seq library preparation from SHY5Y and U1 transfected HEK293
SHY5Y cells were seeded in six-well plates at 6 × 105 cells/well in 2 mL 10% FBS, DMEM and incubated for 4 h. Cells were then treated with two biological replicates of HTT-C1 at 24 nM or 100 nM (in 0.1% DMSO), or four biological replicates of vehicle control (DMSO) for 24 h (37 °C, 5% CO2, 100% relative humidity). HEK293 cells were transfected with U1-GA variant minigene construct or mock for 48 h (37 °C, 5% CO2, 100% relative humidity).
Total RNA was extracted using the RNeasy Plus Mini Kit. RNA concentration and quality were assessed using a NanoDrop spectrophotometer (ThermoFisher Scientific). For library preparation and sequencing, mRNA was enriched from ~3 µg of total RNA using oligo(dT) beads. The mRNA was fragmented randomly using fragmentation buffer followed by complementary DNA (cDNA) synthesis using an mRNA template and random hexamers primer. Second-strand synthesis buffer (Illumina), deoxynucleotides, ribonuclease H and DNA polymerase I were added to initiate second-strand synthesis. After a series of terminal repair, A-ligation and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment. RNA libraries were sequenced in a HiSeq sequencer (Illumina).
RNA-Seq analysis of pre-mRNA splicing
RNA sequencing reads were mapped to human genome (hg19) using STAR (version 2.5)34; only uniquely mapped reads (with MAPQ > 10) with <5nt/100nt mismatches and properly paired reads were used. For gene expression analysis, the number of reads in the coding sequence (CDS) region of protein-coding genes and exonic region of non-coding genes were counted and analysed using DESeq235 (Bioconductor). For splicing analysis, all junction reads (read with a gap in alignment indicating splicing) were used, including the ones mapped to unannotated splice sites. Reads were counted for different exons (for cassette exon [CE]) or exonic regions (for A5′ss or A3′ss). For each splicing event, a percent-spliced-in (PSI) value was calculated using the percent of average read number supporting the inclusion among all reads supporting either the inclusion or the exclusion. A minimum of 20 for the denominator of PSI calculation was required. Otherwise, a ‘NA’ value would be generated. PSI values for biological replicates were averaged, and the PSI difference between the two treatment groups was calculated. For a statistical test, a 2 × 2 read counts table was made for each event, with rows for reads supporting inclusion or exclusion, and columns for the two comparing sample groups (biological replicates were combined). Fisher’s exact test was used for the statistical tests. PSI change of >20% (or < −20%) and P < 0.001 was used to select splicing events being regulated by the treatment. PSI calculation, Fisher’s exact test, k-mer analysis and statistical analysis were performed using R (3.5.1).
K-mer analysis
For comparing sequence difference of a particular region for two groups of exons (e.g., Inc vs. NC) we compared the k-mer (k = 4–6) frequencies of the two groups by Fisher’s Exact Test (one k-mer vs. all other k-mers, Group 1 vs. Group 2). The resulting P value was converted to a significance score ([SS] = −S*log10 P value), in which S is the sign indicating enrichment (1) or depletion (−1) of the k-mer in Group 1.
Sequence logo
Sequence logos were generated using WebLogo36 (University of California, Berkeley).
Genome-wide identification of putative GA-psiExons
We searched the human genome with “AGAgtaag” sequences (potential 5′ss sequence responding to the compound) within introns of ReSeq annotated genes. Then 3′ss sequences were scanned upstreaming of the “AGAgtaag” sequence using MaxEntScan33. A putative GA-psiExon is defined as an unannotated exon with length between 6 and 200nt and 3′ss score >2.3.
Minigene constructs
HEK293 cells transfected with minigene constructs were treated with varying concentrations of test compounds in a final concentration 0.05% DMSO and incubated for 24 h. Total RNA was isolated from the cells using the RNeasy Plus Mini Kit (Qiagen) and RNA concentration and quality were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). To determine splicing changes, cDNA was synthesised using the iScript™ cDNA synthesis kit (Bio-Rad Laboratories) according to the manufacturer’s instructions. Endpoint PCRs were set up using Platinum™ PCR SuperMix High Fidelity (Invitrogen) and the resulting PCR products were separated on 2% E-gels (Invitrogen). Primers (designed by PTC Therapeutics, supplied by Invitrogen) were directed against common sequences in the minigene constructs: T7 Forward: 5′-TAATACGACTCACTATAGGG-3′; BGH Reverse, 5′-TAGAAGGCACAGTCGAGG-3′.
Animal studies
All in-life animal procedures were performed in a laboratory certified by the American Association for the Accreditation of Laboratory Animal Care (AAALAC) with approval from the Institutional Care and Animal Use Committee. BACHD20 and Hu97/1824 mice were used, and the genotype of each BACHD animal was confirmed by an in-house PCR assay prior to enrolment in the study.
Quantification of HTT protein in animal tissues
Test mice were euthanised, and brain, muscle (quadriceps), other peripheral tissues and blood samples were harvested 2 h after the last dose on Day 20. Prior to analysis, crude total protein from brain and peripheral tissue samples were prepared by sample lysis in MSD assay buffer 1 (MSD) with Complete™ Protease Inhibitor Cocktail added (Roche Diagnostics). Tissues were then homogenised using TissueLyser II (Qiagen), plus a 5 mm stainless steel bead. The lysate was clarified by centrifugation at 16,000 × g for 20 min at 4 °C, and the total protein concentration quantified with the Pierce™ BCA Protein Assay Kit (Thermo Scientific), according to the manufacturer’s instructions. Whole blood was collected by cardiac puncture into EDTA collection tubes. An aliquot (100─200 µL) was added to 1.5 mL of eBioscience™ 1v Red Blood Cell Lysis Buffer (Thermo Fisher) and mixed well for 5 min before collecting the white blood cells (WBC) by centrifugation at 400 × g. The supernatant was discarded, and the pellet of WBCs was frozen in liquid nitrogen and stored at −70 °C. Brain and peripheral sample lysates were analysed for hHTT and KRAS protein expression using the ECL protein assay (described previously in Methods); using the same method WBC samples were also analysed for hHTT expression but not KRAS.
Each tissue sample was tested in duplicate using the ECL protein assay and the average hHTT and KRAS readouts were calculated. The ratio of the mean hHTT signal to the mean KRAS signal (×1000) was determined for each test animal. The hHTT/KRAS ratio grand mean for the vehicle group of five test animals was calculated, and the fold change relative to the vehicle grand mean was determined for each test animal in each group. Percent hHTT (%hHTT) lowering normalised to KRAS was determined for each test animal by subtracting the fold change from one and multiplying the difference by 100. Each experiment was performed twice yielding ten %hHTT-lowering values for each treatment group. For each treatment group, the mean %hHTT lowering plus the standard error of the mean was plotted as a bar graph. The %hHTT lowering in WBC samples was determined without KRAS using the grand hHTT vehicle mean, instead of the grand hHTT/KRAS ratio vehicle mean.
In vivo pharmacokinetic studies
Oral pharmacokinetics (PK) of compounds were evaluated in wt littermates from the BACHD colony (FVB background). Mice were treated with test compounds (10 mg/kg) by oral gavage in 0.5% hydroxypropylmethyl cellulose (HPMC) with 0.1% Tween 80. Blood was collected by terminal cardiac puncture at specified time points (three mice per time point) and centrifuged to generate plasma. Brain tissue was collected at the time of blood collection and homogenised in water. Protein was precipitated from plasma and brain homogenates with acetonitrile, methanol mixture (5:1, v/v) containing an internal standard that is a close analogue of the test compounds. The mixture was filtered through an EMD Millipore MultiScreen™ Solvinert Filter Plate (MSRLN04, Millipore, Burlington, MA). Calibration standards were prepared in the same matrix and processed with the testing samples. Filtrates were analysed using an Acquity ultra performance liquid chromatography (UPLC) system (Waters Corporation) in tandem with Xevo TQ-s Spectrometer (Waters Corporation). Samples were injected on to a Waters UPLC Acquity BEH C18 Column (2.1 × 50 mm, 1.7 μm) maintained at 50 °C. The injection volume was 3 µL and the mobile phase flow rate was 0.45 mL/min. The mobile phase consisted of two solvents: a) 0.1% formic acid in water and b) 0.1% formic acid in acetonitrile. The initial mobile phase started with 5% solvent B for 0.4 min, which was changed to 98% solvent B over 0.8 min with linear gradients, and then maintained at 95% solvent B for another 0.4 min. The drug concentrations were acquired and processed with MassLynx 4.1 software. PK parameters were estimated using the non-compartment method within Phoenix® WinNonlin® Build 8.1 (Certara USA, Inc., Princeton, NJ).
In vivo pharmacodynamic studies
BACHD: pharmacodynamic (PD) evaluations were performed in BACHD mice aged 6−10 weeks. Compound or vehicle (HPMC/0.1% Tween 80) was administered to BACHD mice (five female mice per group) once daily for 21 doses (QD×21) by oral gavage; dosing volumes were 10 mL/kg. Each animal was regularly observed for mortality or signs of pain, distress or overt toxicity, and findings were recorded. Body weights were recorded at the start, and at least once a week, during the course of the study. Tissue samples were obtained and prepared for ECL protein assay analysis (described previously in Methods) from each animal.
Hu97/18: both sexes of 2–4-month-old Hu97/18 mice were used. Mice were maintained under a 12-hour light:12-hour dark cycle in a clean facility with free access to food and water. Experiments were performed with the approval of the Institute Animal Care and Use Committee of the University of Central Florida. Mice were treated with vehicle control or 2, 6 or 12 mg/kg of compound daily by oral gavage for 21 consecutive days. Mice were weighed 3x weekly and observed daily for general health and neurological signs, including gait, head tilt and circling. No adverse events were observed, and no mice were removed from the study.
Hu97/18 terminal tissue and sample collection
Mice were anaesthetised with Avertin (2,2,2-tribromoethanol, Sigma Aldrich, catalogue # T48402) and secured in a stereotaxic frame (Stoelting). The ear bars were raised and the nose piece used to position the mice in a manner that would allow for a near 90° tilt of the head to access the cisterna magna. A 1 cm2 section of dorsal neck skin was removed, and muscle layers were completely dissected away to expose the cisterna magna, which was then cleaned with PBS and 70% ethanol and dried using compressed air. A 50cc Hamilton® syringe with point style 2 and a 12o bevel was then lowered carefully into the cisterna magna. CSF was slowly withdrawn at a rate of 10 μl/min using an UltraMicroPump with a Micro4 controller (World Precision Instruments). CSF samples were collected in pre-chilled tubes, centrifuged, then flash frozen in liquid nitrogen prior to storage at −80 °C.
Whole blood was then collected by cardiac puncture into EDTA-coated tubes and divided into three aliquots. One was immediately snap frozen, while plasma was isolated from another and crude peripheral blood mononuclear cells from the third. Mice were then decapitated, and the brain removed and placed in ice for ~1 min to increase tissue rigidity. During this interval, liver, heart and quadriceps muscle were isolated and snap frozen. Brains were then micro-dissected into cortex, hippocampus, striatum, cerebellum, and midbrain/brain stem.
Immunoprecipitation and flow cytometry mtHTT quantification
Approximately 10,000 5-μm carboxylate-modified latex beads (Invitrogen, catalogue # C37255) were coupled with capture antibody, HDB4E10 anti-HTT, in 50 μl of NP40 lysis buffer (150 mM NaCl, 50 mM Tris pH 7.4, Halt phosphatase (Thermo Scientific, catalogue # 78420) and Halt protease inhibitor cocktails (Thermo Scientific, catalogue # 78429), 2 mM sodium orthovanadate, 10 mM sodium fluoride NaF, 10 mM Iodoacetamide, and 1% NP40). Capture antibody coupled beads were then combined with 10 μl of CSF, or 20 μl of plasma in triplicate in a 96-well V-bottom plate (Thermo Scientific, catalogue # 249944), brought to a total volume of 50 μl in NP40 lysis buffer, mixed well, and incubated overnight at 4 °C. The next day, the plate was spun down for 1 min at 650 RCF and supernatant was removed. Beads were washed three times in immunoprecipitation and flow cytometry (IP-FCM) wash buffer (100 mM NaCl, 50 mM Tris pH 7.4, 1% BSA, 0.01% sodium azide). MW1 anti-expanded polyglutamine probe antibody was biotinylated using EZ-Link Sulfo-NHS-Biotin (Thermo Scientific, catalogue # 21217), and 50 μl of the diluted antibody was incubated with the HDB4E10 beads bound to mtHTT for 2 h at 4 °C. Beads were washed three times with 200 μl of IP-FCM wash buffer. Streptavidin–phycoerythrin (PE) (BD Biosciences, catalogue # 554061) was prepared at 1:200 and 50 μl added to each well and incubated at RT, protected from light, for 30 min. Beads were washed three times with 200 μl of IP-FCM buffer, resuspended in 200 μl of IP-FCM wash buffer, and fluorescence intensity of ~2000 beads per sample, HDB4E10/MW1 mtHTT bead complexes, was measured using an Acuri C6 flow cytometer (BD Biosciences). Median fluorescent intensity of PE was measured for each sample to determine relative mtHTT protein levels.
MDCK-MDR1 efflux assay
The MDR1 efflux assay was conducted at Absorption System LLC (Exton, PA). In brief, MDCK-MDR1 and MDCK-wt cell monolayers were grown to confluence on collagen-coated, microporous membranes in 12-well assay plates (Thermofisher). Compound solutions (10 μM) in permeability assay buffer (Hanks’ balanced salt solution [HBSS], 10 mM HEPES, 15 mM glucose; pH of 7.4) were placed in the donor chamber. The receiver chamber was filled with assay buffer plus 1% BSA. Cell monolayers were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37 °C (5% CO2, 100% relative humidity). Sampling from the donor chambers was performed at 0 and 1 hr; and from the receiver chambers at 1 hr. Each determination was performed in duplicate. The flux of lucifer yellow was also measured post-experimentally for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by liquid chromatography-tandem mass spectrometry using electrospray ionisation. The apparent permeability (Papp) and percent recovery was determined using the following equation:
({P}_{app}=({{{{{rm{d}}}}}}{C}_{{{{{{rm{r}}}}}}}/{{{{{rm{dt}}}}}})ast {V}_{{{{{{rm{r}}}}}}}/(Atimes {C}_{0}))
dCr/dt represents the slope of the cumulative receiver concentration vs. time in μM/s; Vr is the volume of the receiver compartment (cm3); Vd is the volume of the donor compartment in (cm3); A is the area of the insert (1.13 cm2 for 12-well); C0 is the average measured concentration of the donor chamber at time zero in μM; net efflux ratio is defined as Papp(B-to-A)−Papp(A-to-B).
Unbound brain partition coefficient (K
p,uu)
The unbound brain partition coefficient (Kp,uu) is defined as the ratio between unbound brain-free drug concentration and unbound plasma concentration. It was calculated using the following equation:
({K}_{{{{{{rm{p}}}}}},{{{{{rm{uu}}}}}}}={C}_{{{{{{rm{brain}}}}}}}ast {f}_{{{{{{rm{u}}}}}},{{{{{rm{b}}}}}}}/({C}_{{{{{{rm{plasma}}}}}}}ast {f}_{{{{{{rm{u}}}}}},{{{{{rm{p}}}}}}}))
Cbrain and Cplasma represent the compound concentrations in brain and plasma, respectively. fu,b and fu,p are the unbound fraction of each testing article in brain and plasma, respectively. Both fu,b and fu,p were determined in vitro using the Pierce device for rapid equilibrium dialysis at Absorption System LLC (Exton, PA).
Kp,uu was calculated individually for each animal from multiple mouse PK studies and the average values are reported here.
Quantification of human HTT mRNA in animal brain
One group of transgenic BACHD mice in the study was orally administered a single dose of HTT-D3 at 10 mg/kg. One group (three mice) was administered vehicle alone on the same schedule. Dosing volumes were 10 mL/kg based on individual mouse weights. Dosing solutions were prepared once as the free base HTT-D3 dissolved in a vehicle comprising 0.5% HPMC and 0.1% TWEEN® 80 and stored at ambient temperature. Samples of brain tissues were obtained at 2, 4, and 8 h post HTT-D3 dosing. Samples were taken from the vehicle control mice at 2-h post vehicle dosing. Brain samples for each time point were obtained from three mice per group and total RNAs were prepared for analysis. Total RNAs from brain tissues were prepared following sample homogenisation and lysis in QIAzol Lysis Reagent using TissueLyser and the RNeasy Lipid Tissue Mini Kit (Qiagen #74804, Germantown, MD) according to the instructions provided in the manufacturer’s kit.
Quantitative RT-qPCR was performed on ~50 ng of total RNA using AgPath-ID™ One-Step RT-qPCR Reagents (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions using the custom TaqMan gene expression assays detailed in Supplementary Table 3.
An RT-qPCR reaction mixture of primers and probe sets for HTT and GAPDH was prepared according to Supplementary Table 4.
A volume of 2 μL of the RNA preps was transferred from each well to the Armadillo 384-well PCR plate containing 8 μL/well of the RT-qPCR reaction mixture that was prepared as detailed in Supplementary Table 4. The plate was then sealed with MicroAmp™ Optical Adhesive Film and placed in the C1000 thermocycler. The RT-qPCR was carried out at the following temperatures for the indicated times: Step 1: 48 °C (30 min); Step 2: 95 °C (10 min); Step 3: 95 °C (15 s); Step 4: 60 °C (1 min); then, repeated Steps 3 and 4 for a total of 40 cycles.
The amplification efficiency was calculated from the slope of the PCR amplification curve for HTT and GAPDH individually. The abundances of HTT mRNA and GAPDH mRNA were then calculated as (1 + E)−Ct, where Cycle threshold (Ct) is the threshold value for each amplicon. The abundance of HTT mRNA was normalised to GAPDH abundance. The normalised HTT mRNA level was then used to calculate the percent splicing in the HTT-D3-dosed group compared to the vehicle-treated group using a modified 2−ΔΔCT method37.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
