Phosphoramidite

hsiRNA design. We designed and synthesized a panel of 94 hsiRNA compounds (Supplementary Table S1) targeting the human huntingtin gene. These sequences span the gene and were selected to comply with standard siRNA design parameters^{24 (link)} including assessment of GC content, specificity and low seed compliment frequency,^{55 (link)} elimination of sequences containing miRNA seeds, and examination of thermodynamic bias.^{56 (link),57 (link)}Oligonucleotide synthesis, deprotection, and purification. Oligonucleotides were synthesized using standard phosphoramidite, solid-phase synthesis conditions on a 0.2–1 µmole scale using a MerMade 12 (BioAutomation, Irving, TX) and Expedite DNA/RNA synthesizer. Oligonucleotides with unmodified 3′ ends were synthesized on controlled pore glass (CPG) functionalized with long-chain alkyl amine and a Unylinker terminus (Chemgenes, Wilmington, MA). Oligonucleotides with 3′-cholesterol modifications were synthesized on modified solid support (Chemgenes). Phosphoramidite solutions were prepared at 0.15 mol/l in acetonitrile for 2′-TBDMS, 2′-O-methyl (Chemgenes), and Cy3 modifications or 0.13 mol/l for 2′-fluoro (BioAutomation) modifications. Phosphoramidites were activated in 0.25 mol/l 4,5-dicyanoimidazole in acetonitrile. Detritylation was performed in 3% dichloroacetic acid in dichloromethane for 80 seconds. Capping was performed in 16% N-methylimidazole in tetrahydrofuran and acetic anhydride:pyridine:tetrahydrofuran, (1:2:2, v/v/v) for 15 seconds. Oxidation was performed using 0.1 mol/l iodine in pyridine:water:tetrahydrofuran (1:2:10, v/v/v).
The CPG was removed from the solid-phase column and placed in a polypropylene screw cap vial. Dimethylsulfoxide (100 µl) and 40% methylamine (250 µl) are added directly to the CPG and shaken gently at 65 °C for exactly 16 minutes. The vial was cooled on dry ice before the cap was removed. The supernatant was transferred to another polypropylene screw cap vial, and the CPG was rinsed with two 150 µl portions of dimethylsulfoxide, which were combined with original supernatant. Oligonucleotides without 2′-TBDMS-protecting groups were lyophilized. Oligonucleotides with 2′-TBDMS-protecting groups were desilylated by adding 375 µl triethylamine trihydrofluoride (~1.5 volumes relative to 40% methylamine) and incubated for exactly 16 minutes at 65 °C with gentle shaking. Samples were quenched by transferring to a 15 ml conical tube containing 2 ml of 2 mol/l triethylammonium acetate buffer (pH 7.0). The sample was stored at −80 °C until high-performance liquid chromatography purification.
Oligonucleotides were purified by reverse-phase high-performance liquid chromatography on a Hamilton PRP-C18 column (21 × 150 mm) using an Agilent Prostar 325 high-performance liquid chromatography (Agilent, Santa Clara, CA). Buffer A 0.05 mol/l tetraethylammonium acetate with 5% acetonitrile, Buffer B 100% acetonitrile, with a gradient of 0% B to 35% B over 15 minutes at 30 ml/minutes. Purified oligonucleotides were lyophilized to dryness, reconstituted in water, and passed over a Hi-Trap cation exchange column to exchange the tetraethylammonium counter-ion with sodium.
Cell culture. HeLa cells (ATCC, Manassas, VA; #CCL-2) were maintained in Dulbecco's Modified Eagle's Medium (Cellgro, Corning, NY; #10-013CV) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA; #26140) and 100 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA; #15140) and grown at 37 °C and 5% CO₂. Cells were split every 2 to 5 days and discarded after 15 passages.
Preparation of primary neurons. Primary cortical neurons were obtained from FVB/NJ mouse embryos at embryonic day 15.5. Pregnant FVB/NJ females were anesthetized by intraperitoneal injection of 250 mg Avertin (Sigma, St Louis, MO; #T48402) per kg weight, followed by cervical dislocation. Embryos were removed and transferred into a Petri dish with ice-cold Dulbecco's Modified Eagle's Medium/F12 medium (Invitrogen; #11320). Brains were removed, and meninges carefully detached. Cortices were isolated and transferred into a 1.5-ml tube with prewarmed papain solution for 25 minutes at 37 °C, 5% CO₂, to dissolve tissue. Papain solution was prepared by suspending DNase I (Worthington, Lakewood, NJ; #54M15168) in 0.5 ml Hibernate E medium (Brainbits, Springfield, IL; #HE), and transferring 0.25 ml DNase I solution to papain (Worthington, Lakewood, NJ; #54N15251) dissolved in 2 ml Hibernate E medium and 1 ml Earle's balanced salt solution (Worthington; #LK003188). After the 25-minute incubation, papain solution was replaced with 1 ml NbActiv4 medium (Brainbits; #Nb4-500) supplemented with 2.5% FBS. Cortices were dissociated by repeated pipetting with a fire-polished, glass, Pasteur pipette. Cortical neurons were counted and plated at 1 × 10⁶ cells per ml.
For live-cell imaging, culture plates were precoated with poly-l-lysine (Sigma; #P4707), and 2 × 10⁵ cells were added to the glass center of each dish. For silencing assays, neurons were plated on 96-well plates precoated with poly-l-lysine (BD BIOCOAT, Corning, NY; #356515) at 1 × 10⁵ cells per well. After overnight incubation at 37 °C, 5% CO₂, an equal volume of NbActiv4 supplemented with anti-mitotics, 0.484 µl/ml of UTP Na₃ (Sigma; #U6625), and 0.2402 µl/ml of FdUMP (Sigma; #F3503), was added to neuronal cultures to prevent growth of nonneuronal cells. Half of the media volume was replaced every 48 hours until the neurons were treated with siRNA. Once the cells were treated, media was not removed, only added. All subsequent media additions contained antimitotics.
Direct delivery (passive uptake) of oligonucleotides. Cells were plated in Dulbecco's Modified Eagle's Medium containing 6% FBS at 10,000 cells per well in 96-well tissue culture plates. hsiRNA was diluted to twice the final concentration in OptiMEM (Gibco; #31985-088), and 50 μl diluted hsiRNA was added to 50 μl of cells, resulting in 3% FBS final. Cells were incubated for 72 hours at 37 °C and 5% CO₂. Based on previous experience, we know that 1.5 µmol/l active hsiRNA supports efficient silencing without toxicity. The primary screen for active Htt siRNAs, therefore, was performed at 1.5 µmol/l compound, which also served as the maximal dose for in vitro dose–response assays.
hsiRNA lipid-mediated delivery. Cells were plated in Dulbecco's Modified Eagle's Medium with 6% FBS at 10,000 cells per well in 96-well tissue culture–treated plates. hsiRNA was diluted to four times the final concentration in OptiMEM, and Lipofectamine RNAiMAX Transfection Reagent (Invitrogen; #13778150) was diluted to four times the final concentration (final = 0.3 µl/25 µl/well). RNAiMAX and hsiRNA solutions were mixed 1:1, and 50 µl of the transfection mixture was added to 50 µl of cells resulting in 3% FBS final. Cells were incubated for 72 hours at 37 °C and 5% CO₂.
mRNA quantification in cells and tissue punches. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix; #QS0011). Cells were lysed in 250 μl diluted lysis mixture composed of 1 part lysis mixture (Affymetrix; #13228), 2 parts H₂O, and 0.167 μg/μl proteinase K (Affymetrix; #QS0103) for 30 minutes at 55 °C. Cell lysates were mixed thoroughly, and 40 μl (~8,000 cells) of each lysate was added per well to a capture plate with 40 μl diluted lysis mixture without proteinase K. Probe sets were diluted as specified in the Affymetrix protocol. For HeLa cells, 20 μl human HTT or PPIB probe set (Affymetrix; #SA-50339, #SA-10003) was added to appropriate wells for a final volume of 100 μl. For primary neurons, 20 μl of mouse Htt or Ppib probe set (Affymetrix; #SB-14150, #SB-10002) was used.
Tissue punches (5 mg) were homogenized in 300 μl of Homogenizing Buffer (Affymetrix; #10642) containing 2 μg/μl proteinase K in 96-well plate format on a QIAGEN TissueLyser II (Qiagen, Valencia, CA; #85300), and 40 μl of each lysate was added to the capture plate. Probe sets were diluted as specified in the Affymetrix protocol, and 60 μl of Htt or Ppib probe set was added to each well of the capture plate for a final volume of 100 μl. Signal was amplified according to the Affymetrix protocol. Luminescence was detected on either a Veritas Luminometer (Promega, Madison, WI; #998–9100) or a Tecan M1000 (Tecan, Morrisville, NC).
Western blot. Cell lysates (25 µg) were separated by SDS–PAGE using 3–8% Tris-acetate gels (Life Technologies, Grand Island, NY; #EA03785BOX) and transferred to nitrocellulose using a TransBlot Turbo apparatus (BioRad, Hercules, CA; #1704155). Blots were blocked in 5% nonfat dry milk (BioRad; #1706404) diluted in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hour at room temperature then incubated in N-terminal antihuntingtin antibody Ab1^{58 (link)} diluted 1:2,000 in blocking solution overnight at 4 °C with agitation. After washing in TBST, blots were incubated in peroxidase-labeled antirabbit IgG (Jackson ImmunoResearch, West Grove, PA; #711035152) diluted in blocking buffer for 1 hour at room temperature, washed in TBST, and proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL; #34080) and Hyperfilm ECL (GE Healthcare, Buckinghamshire, UK; #28906839). Blots were reprobed with anti-β tubulin antibody (Sigma; #T8328) as a loading control. Films were scanned with a flatbed scanner (Epson Perfection V750 Pro; Epson, Long Beach, CA), and densitometry was performed using NIH ImageJ software to determine total intensity of each band. The huntingtin signal was divided by the tubulin signal to normalize to protein content, and percent of untreated control was determined for each set of samples (N = 5).
Live cell imaging. To monitor live cell hsiRNA uptake, cells were plated at a density of 2 × 10⁵ cells per 35-mm glass-bottom dish. Cell nuclei were stained with NucBlue (Life Technologies; #R37605) as indicated by the manufacturer. Imaging was performed in phenol red-free NbActiv4 (Brainbits; #Nb4-500). Cells were treated with 0.5 μmol/l Cy3-labeled hsiRNA, and live cell imaging was performed over time. All live cell confocal images were acquired with a Leica DM IRE2 confocal microscope using 63x oil immersion objective (Buffalo Grove, IL), and images were processed using ImageJ (1.47v) software.
Stereotaxic injections. FVB/NJ mice (50% male and 50% female for each dose group, 6–8 weeks old) were deeply anesthetized with 1.2% Avertin (Sigma; #T48402) and microinjected by stereotactic placement into the right striatum (coordinates relative to bregma: 1.0 mm anterior, 2.0 mm lateral, and 3.0 mm ventral). For both toxicity (DARPP-32 staining) and efficacy studies, mice were injected with either PBS or artificial CSF (2 μl per striata), 12.5 μg of nontargeting hsiRNA (2 μl of 500 µmol/l stock per striata), 25 μg of HTT10150 hsiRNA (2 μl of 1 mmol/l stock per striata), 12.5 μg of HTT10150 hsiRNA (2 μl of 500 μmol/l stock per striata), 6.3 μg of HTT10150 hsiRNA (2 μl of 250 μmol/l stock per striata), or 3.1 μg of HTT10150 hsiRNA (2 μl of 125 μmol/l stock per striata). For toxicity studies, n = 3 mice were injected per group, and for efficacy studies, n = 8 mice were injected per group. Mice were euthanized 5 days post-injection, brains were harvested, and three 300-μm coronal sections were prepared. From each section, a 2-mm punch was taken from each side (injected and noninjected) and placed in RNAlater (Ambion, Carlsbad, CA; #AM7020) for 24 hours at 4 °C. Each punch was processed as an individual sample for Quantigene 2.0 assay analysis (Affymetrix) and averaged for a single animal point. All animal procedures were approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee (protocol number A-2411).
Immunohistochemistry/immunofluorescence. Mice were injected intrastriatally with 12.5 µg of Cy3-labeled hsiRNA. After 24 hours, mice were sacrificed and brains were removed, embedded in paraffin, and sliced into 4-μm sections that were mounted on glass slides. Sections were deparaffinized by incubating in Xylene twice for 8 minutes. Sections were rehydrated in serial ethanol dilutions (100%, 95%, and 80%) for 4 minutes each, and then washed twice for 2 minutes with PBS.
For NeuN staining,^{39 (link),40 (link)} slides were boiled for 5 minutes in antigen retrieval buffer (10 mmol/l Tris/ 1mmol/l EDTA (pH 9.0)), incubated at room temperature for 20 minutes, and then washed for 5 minutes in PBS. Slides were blocked in 5% normal goat serum in PBS containing 0.05% Tween 20 (PBST) for 1 hour and washed once with PBST for 5 minutes. Slides were incubated with primary antibody (Millipore, Taunton, MA; MAB377, 1:1,000 dilution in PBST) for 1 hour and washed three times with PBST for 5 minutes. Slides were then incubated with secondary antibody (Life Technologies; #A11011, 1:1000 dilution in PBST) for 30 minutes in the dark and washed three times with PBST for 5 minutes each. Slides were then counterstained with 250 ng/ml 4',6-diamidino-2-phenylindole (Molecular Probes, Life Technologies; #D3571) in PBS for 1 minute and washed three times with PBS for 1 minute. Slides were mounted with mounting medium and coverslips and dried overnight before imaging on a Leica DM5500 microscope fitted with a DFC365 FX fluorescence camera.
For toxicity studies, injected brains were harvested after 5 days. For microglial activation studies, brains were harvested after 6 hours or 5 days. Extracted, perfused brains were sliced into 40-µm sections on the Leica 2000T Vibratome (Leica Biosystems, Wetzlar, Germany) in ice-cold PBS. Every sixth section was incubated with DARPP-32 (Abcam, Cambridge, UK; #40801; 1:10,000 in PBS) or IBA-1 (Wako; #019-19741; 1:1,000 in PBS) antibody, for a total of nine sections per brain and eight images per section (four per hemisphere). IBA-1 sections were incubated in blocking solution (5% normal goat serum, 1% bovine serum albumin, 0.2% Triton-X-100, and 0.03% hydrogen peroxide in PBS) for 1 hour, and then washed with PBS. Sections were incubated overnight at 4 °C in primary antibody, anti-Iba1 (polyclonal rabbit anti-mouse/human/rat; dilution: 1:1,000 in blocking solution) (Wako; #019-19741). Sections were then stained with goat antirabbit secondary antibody (1:200 dilution) (Vector Laboratories, Burlingame, CA), followed by a PBS wash, the Vectastain ABC Kit (Vector Laboratories), and another PBS wash. IBA-1 was detected with the Metal Enhanced DAB Substrate Kit (Pierce, Rockford, IL). For DARPP32 staining, sections were washed for 3 minutes in 3% hydrogen peroxide, followed by 20 minutes in 0.2% TritonX-100 and 4 hours in 1.5% normal goat serum in PBS. Sections were incubated overnight at 4 °C in DARPP32 primary antibody (1:10,000 dilution) (Abcam; #40801) made up in 1.5% normal goat serum. Secondary antibody and detection steps were conducted as described for IBA-1 staining. DARPP-32 sections were mounted and visualized by light microscopy with 20× objective on a Nikon Eclipse E600 with a Nikon Digital Sight DSRi1 camera (Nikon, Tokyo, Japan). The number of DARPP-32-positive neurons was quantified manually using the cell counter plug-in on ImageJ for tracking. Activated microglia were quantified by morphology of IBA-1-positive cells^{42 (link),43 (link),44 (link),45 (link)} from the same number of sections captured with 40× objective. Counting of both IBA-1- and DARPP-32-positive cells was blinded. Coronal section images were taken with a Coolscan V-ED LS50 35-mm Film Scanner (Nikon, Tokyo, Japan).
Statistical analysis. Data were analyzed using GraphPad Prism 6 software (GraphPad Software, Inc., San Diego, CA). Concentration-dependent IC₅₀ curves were fitted using a log(inhibitor) versus response–variable slope (four parameters). The lower limit of the curve was set at zero, and the upper limit of the curve was set at 100. For each independent mouse experiment, the level of knockdown at each dose was normalized to the mean of the control group (the noninjected side of the PBS or artificial CSF groups). In vivo data were analyzed using a two-way repeated-measures analysis of variance with Tukey's multiple comparisons test for dose and side of brain. Differences in all comparisons were considered significant at P values less than 0.05 compared with the NTC- injected group. P values reported represent significance of the entire dose group relative to NTC and are not specific to the ipsilateral or contralateral side. For microglial activation, significance was calculated using a parametric, unpaired, two-tailed t-test for comparison between dose groups, and paired t-test for comparison between ipsilateral and contralateral hemispheres within the same dose group.
SUPPLEMENTARY MATERIALFigure S1. Active hsiRNAs silence huntingtin mRNA in a concentration dependent manner in HeLa cells.
Figure S2. HTT10150 does not affect primary cortical neuron viability.
Figure S3. HTT10150 causes a slight increase in total resting microglia 5 days post injection.
Figure S4. HTT10150 shows limited toxicity at the site of injection at the 25 µg dose.
Table S1. Detailed sequence, chemical modification patterns, and efficacy of hsiRNAs.

[γ-³²P]-ATP was from Perkin Elmer Health Sciences Inc. (Shelton, CT). dNTPs were purchased from New England Biolabs (Beverly, MA). Oligonucleotides were either synthesized on an ABI 394 oligonucleotide synthesizer at JHU or obtained from Integrated DNA Technologies (Coralville, IA). Fapy•dG-containing oligonucleotides were synthesized as described previously (21 (link)), purified by 20% denaturing polyacrylamide gel electrophoresis and characterized by MALDI-TOF MS (Supplementary Figures S2 and S3). PC Spacer phosphoramidite for 5′-phosphorylation, 5′-cyanoethyl phosphoramidites for thymidine (T), N,N-dimethylformamidine-2′-deoxyguanosine (dG^dmf), as well as other reagents required for oligonucleotide synthesis were purchased from GLEN Research (Sterling, VA). N-Acetyl 2′-deoxycytidine 5′-cyanoethyl phosphoramidite (dC^Ac) and Universal UnyLinker™ Support were purchased from Chemgenes (Wilmington, MA). 5′-cyanoethyl phosphoramidite of N-Pac-2′-deoxyadenosine was prepared as previously described (22 (link)). Disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate for methyl deprotection was prepared as previously described (38 (link)).

Oligonucleotides were synthesized using modified (2'-F, 2'-o-Me, locked nucleic acid [LNA]) phosphoramidites with standard protecting groups. Phosphoramidite solid-phase synthesis was done on a MerMade12 (BioAutomation) using modified protocols. Unconjugated oligonucleotides were synthesized on CPG functionalized with a long-chain alkyl amine (LCAA) and unylinker terminus (Chemgenes). Cholesterol CPG was purchased from Chemgenes (Wilmington MA) Product # N-9166–05. GalNAc-conjugated oligonucleotides were grown on custom 3′-GalNAc-CPG (23 (link)); divalent oligonucleotides (DIOs) were synthesized on modified solid support (5 (link)), and VP phosphoramidite was synthesized as described. Phosphoramidites were prepared at 0.1 M in anhydrous ACN, with added dry 15% DMF in the 2′-OMe U amidite. 5-(Benzylthio)-1H-tetrazole (BTT) was used as the activator at 0.25 M. Detritylations were performed using 3% trichloroacetic acid in DCM. Capping was done with non-tetrahydrofuran-containing reagents CAP A, 20% n-methylimidazole in ACN and CAP B, 20% acetic anhydride (Ac₂O) and 30% 2,6-lutidine in ACN (synthesis reagents were purchased at AIC). Sulfurization was performed with 0.1 M solution of 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine (ChemGenes) for 3 min. Phosphoramidite coupling times were 3 min for all amidites used.