Care and use of laboratory animals
All studies were conducted using protocols consistent with local, state and federal regulations, as applicable, and approved by the Institutional Animal Care and Use Committee (IACUC) at Alnylam Pharmaceuticals, Charles River Laboratories (CRL), or LabCorp (formerly Covance, Inc.), as applicable.
Oligonucleotide synthesis
All oligonucleotides were synthesized on an MerMade 192 or MerMade 12 synthesizer according to previously published protocols30,43. 5'-O-(4,4'-Dimethoxytrityl)-2'-deoxy-2'-fluoro- and 5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite monomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine and 2-N-isobutyrylguanosine were purchased commercially and (S)-GNA phosphoramidites were synthesized according to previously published protocols44-46. 2'-O-C16 phosphoramidites47 were used at a concentration of 100-150 mM in acetonitrile with no other changes to synthetic protocols. Phosphorothioate linkages were introduced by sulfurization of phosphite linkages utilizing 0.1 M 3-((N,N-dimethyl-aminomethylidene)amino)-3H-1, 2, 4-dithiazole-5-thione (DDTT) in pyridine. After completion of the solid-phase synthesis, the solid support was incubated in a sealed container with aqueous ammonium hydroxide (28-30%) with added 5% diethylamine by volume, with shaking overnight at 35° C, following a procedure optimized for 5’-(E)-vinyl phosphonate oligonucleotide deprotection48. The oligonucleotide was filtered to remove the support with 5x volume of water and analyzed by LC-MS and ion-exchange HPLC.
After deprotection and crude quality confirmation, ion-exchange HPLC purification was performed. Purification buffer A consisted of 20 mM sodium phosphate, 15% ACN, pH 8.5. Buffer B was the same composition with an additional 1 M sodium bromide. TSKgel Super Q-5PW (20) anion exchange resin (Tosoh Corporation, 0018546) was used for purification, and a general purification gradient of 15% to 48% in about 20 column volumes was applied. Fractions were analyzed by ion-exchange analysis using a Dionex DNAPac PA200 ion-exchange analytical column, 4mm x 250mm (ThermoFisher, 063000) at room temperature. Buffer A consisted of 20 mM sodium phosphate, 15% acetonitrile, pH 12. Buffer B was identical with additional 1 M sodium bromide. A gradient of 30% to 50% over 12 min with a flow rate of 1 mL/min was used to analyze fractions. Fractions with greater than 85% purity were pooled, concentrated, and desalted over size exclusion columns (GE Healthcare,17-5087-01) with a flow rate of 10 mL/min. All oligonucleotides were purified and desalted, and further annealed to form siRNAs duplexes as previously described12. All siRNAs were synthesized by Alnylam Pharmaceuticals (Cambridge, MA).
The identities and purities of all oligonucleotides were confirmed using ESI-LC/MS and IEX HPLC, respectively.
In vitro screening
Rat RPE-J cells (ATCC, CRL-2240) were transfected by adding 0.1 µL of RNAiMAX (Invitrogen, 13778) diluted in 4.9 µL of Opti-MEM to 5 µL of siRNA in a 384-well plate. After a 15 min room temperature incubation, 40 µL of media containing ~5 x103 cells was added to the wells. Cells were incubated for 24 hours, and RNA was isolated using an automated protocol on a BioTek-EL406 platform using Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen, 61012) according to the manufacturer’s instructions. Briefly, 70 µL of Lysis/Binding Buffer and 10 µL of Lysis Buffer containing 3 µL of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature. Bead-bound RNA was then washed two times with 150 µL Wash Buffer A and once with Wash Buffer B utilizing magnetic beads. RNA was eluted with 150 µL Elution Buffer.
Complementary DNA (cDNA) synthesis was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368813) according to the manufacturer’s instructions. Briefly, 10 µL of a master mix containing 1 µL 10X Buffer, 0.4 µL 25X dNTPs, 1 µL 10x random primers, 0.5 µL Reverse Transcriptase, 0.5 µL RNase inhibitor and 6.6 µL water per reaction was added to isolated RNA. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 min at room temperature, followed by a 2 hour incubation at 37 °C. 2 µL of cDNA were added to a master mix containing 0.5 µL of rat GAPDH TaqMan probe (Thermo Fisher, 4352338E), 0.5 µL rat SOD1 TaqMan probe (Thermo Fisher, Rn00566938_m1), and 5 µL Lightcycler 480 probe master mix (Roche, 04887301001) per well in a 384-well plate (Roche, 04887301001). Real time quantitative PCR (RT-qPCR) was performed in a LightCycler480 Real Time PCR system (Roche). qPCR data were analyzed using the ΔΔCt method.
Rat IT studies
siRNAs formulated at up to 30 mg/mL in artificial cerebrospinal fluid (aCSF) were administered as 30 µL IT injections by lumbar puncture in the dorsal region of the spine between the L3-L5 vertebral space to male Sprague Dawley rats. At a minimum of 30 minutes prior to surgery, rats were subcutaneously administered 1 mg/kg meloxicam and 0.1 mg/kg buprenorphine. Following anesthesia with isoflurane, rats were placed on a warm heating pad, treated with eye lubricant, and the IT injection site was shaved and disinfected, and an incision was made to expose the spinal column. siRNA was administered with an insulin syringe. Proper placement of the needle was confirmed via CSF backflow in the hub of the needle. Once siRNA administration has been completed, gentle constant pressure on the plunger was maintained for 30 seconds, the incision was sutured and secured with tissue glue, and the rats were placed in sternal recumbency on a heating pad until recovery.
NHP IT studies
siRNAs formulated at 30 mg/mL in aCSF were administered as 2 mL IT injections over 3 minutes by lumbar puncture in the dorsal region of the spine between the L2-L3, L3-L4, L4-L5, or L5-L6 vertebral space of cynomolgus monkeys. Anesthesia was induced with dexmedetomidine and ketamine, and was maintained with supplemental isoflurane as needed. Proper placement of the needle was confirmed via CSF backflow in the hub of the needle. A total volume of 1 mL of CSF was removed prior to dosing, and 0.3 mL aCSF flush was performed following siRNA administration. After completion of the flush, the syringe and needle were removed, and pressure was be applied to injection site for at least 30 seconds, and the animals were allowed to recover. Dexmedetomidine was reversed with atipamezole, as necessary. Serial CSF collections were performed from cisterna magna following procedural anesthesia induced with dexmedetomidine and ketamine and maintained with supplemental isoflurane as needed.
NHP IVT studies
A topical antibiotic (tobramycin) was applied to both eyes twice on the day before and twice on the day after IVT injection to cynomolgus monkeys. The animals received an intramuscular injection of a sedative cocktail (ketamine 5 mg/kg; dexmetedomidine 0.01 mg/kg) followed by isoflurane/oxygen mix through a mask, if deemed necessary to maintain anesthesia. Either 1x phosphate-buffered saline (PBS) or siRNA formulated in 1x PBS were administered in both eyes by IVT injection using a 1 mL syringe and a 30-gauge, ½-inch needle. The dose volume administered was 50 μL/eye. Intravitreal injections were performed by a board-certified veterinary ophthalmologist. Following completion of the dosing procedure, animals received an intramuscular injection of 0.1 mg/kg atipamezole, a reversal agent for dexmetedomidine, as considered necessary. The conjunctivae were flushed with diluted benzalkonium chloride (Zephiran™). Mydriatic drops were be applied to each eye needed.
Mouse IN studies
Female C57BL/6 mice (8-10 weeks of age) were dosed via intranasal instillation (50 μL total, 25 μL/nostril) at the indicated siRNA concentration diluted in 1x PBS and were sacrificed at the indicated day.
RNA extraction and RT-qPCR
For the extraction of RNA, powdered tissues (~10 mg) were resuspended in 700 µL QIAzol and homogenized by vigorous pipetting. Alternatively, two 5 mm steel grinding balls were added to each sample, followed by homogenization at 25/s for 1 min at 4 °C using TissueLyser II (Qiagen, 85300). Samples were incubated at room temperature for 5 minutes followed by addition of 140 µL chloroform. Samples were mixed by shaking, followed by a 10-minute incubation at room temperature. Samples were spun at 6,000 x g for 15 minutes at 4 °C, the supernatant was transferred to a new tube, and 1.5 volumes of 100% ethanol were added. Samples were then purified using a miRNeasy Kit (Qiagen, 217061) according to the manufacturer’s instructions. The RNA was eluted from miRNeasy columns with 50-60 µL RNAse-free water and quantified on a Nanodrop (Thermo Fisher).
Complementary DNA (cDNA) synthesis was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368813) according to the manufacturer’s instructions. Briefly, 10 µL of a master mix containing 2 µL 10X Buffer, 0.8 µL 25X dNTPs, 2 µL 10X Random Primers, 1 µL MultiScribeTM Reverse Transcriptase, 1 µL RNase Inhibitor, and 3.2 µL of nuclease-free water per reaction was added to 10 µL of the isolated RNA. Plates were incubated in a Thermal Cycler programed for four steps: Step 1 – 25 °C for 10 minutes, Step 2 – 37 °C for 120 minutes, Step 3 – 85 °C for 5 minutes, and Step 4 – hold at 4 °C. qPCR reactions were performed using gene specific TaqMan assays for rat SOD1 (Thermo Fisher, Rn00566938_m1), rat PPIB (Thermo Fisher, Rn03302274_m1), mouse SOD1 (IDT, Mm.PT.58.12368303), mouse GAPDH (Thermo Fisher, Mm99999915_g1), NHP APP (Thermo Fisher, Mf01552283_m1), NHP TTR (Thermo Fisher, Mf02799963_m1), NHP PPIB (Thermo Fisher, Mf02802985_m1), or NHP GAPDH (forward primer: 5’-GCATCCTGGGCTACACTGA-3’, reverse primer: 5’-TGGGTGTCGCTGTTGAAGTC-3’, probe: 5’HEX-CCAGGTGGTCTCCTCC-3’BHQ-1). qPCR reactions were performed in a Roche LightCycler 480 using LightCycler 480 Probes Master Mix (Roche, 04707494001). qPCR data were analyzed using the ΔΔCt method.
Protein analyses
For sAPP protein analysis in NHP CSF, a V-PLEX panel for sAPPα and sAPPβ (Meso Scale Discovery, K15120E) was used according to the manufacturer’s instructions. Briefly, CSF samples were thawed on ice for ~1 hour, followed by centrifugation at 2,000 x g for 5 minutes at 4° C to pellet debris. The resultant supernatant was diluted 1:8 in assay diluent (1% Blocker A in 1X Tris Wash Buffer). Plates were blocked in 3% Blocker A in 1X Tris Wash Buffer for at room temperature for 1 hour on a plate shaker at 600 rpm, and washed 3X with 250 µL 1X Tris Wash Buffer on a ELx405 Biotek plate washer. Samples and calibrators were added to plates and incubated at room temperature for 1 hour on a plate shaker at 600 rpm. Plates were washed 3X with 250 µL 1X Tris Wash Buffer, followed by addition of 1X SULFO-TAG anti-APP detection antibody, and incubated at room temperature for 1 hour on a plate shaker at 600 rpm. Plates were washed 3X with 250 µL 1X Tris Wash Buffer, followed by addition of 150 µL 1X Read Buffer T. Plates were incubated at room temperature for ~10 minutes before reading on a MSD SECTOR Imager Instrument (Meso Scale Discovery).
For TTR protein analysis in NHP aqueous humor, an in-house sandwich ELISA was developed. 96-Well Nunc Maxisorp plates (Invitrogen, 44-2404-21) were coated overnight at 4 °C with Rabbit anti-human TTR pAb (Dako, A0002) at a final concentration of 5.63 µg/ml prepared in 50 mM Carbonate/Bicarbonate buffer. The following day, plates were washed 5X with 300 µL Tris Buffer containing 0.05% Tween-20 (TBS-T) on a BioTek plate washer. Plates were blocked with 150 µL/well of 1X Powerblock (Biogenex Labs, HK0855K) for 2 hours at room temperature and washed 5X with 300 µL TBS-T. An in-house Cynomolgus TTR standard was used to prepare the highest standard at 59.5 ng/ml, followed by a 2.5-fold dilution series in 1X Powerblock to generate an 8-point standard curve. Aqueous humor samples were tested at a 1:600 dilution in 1X Powerblock. Standards and samples were added to the plate, 100 µL/well, in duplicate and incubated for 2 hours at room temperature with shaking at 600 rpm. Plates were washed 5X with 300 µL TBS-T, followed by addition of secondary antibody, anti-hTTR pAb (Abcam, ab9015), at a final concentration of 4 ug/ml prepared in 1X PowerBlock. Plates were incubated for 1 hour at room temperature with shaking at 600 rpm. Plates were washed 5X with 300 µL TBS-T, and the detection antibody, Donkey anti-sheep pAb Alkaline-Phosphatase, was applied at a final concentration of 1.4 ug/ml in 1X Powerblock (Sigma, A5187). Plates were incubated for 1 hour at room temperature with shaking at 600 rpm. Plates were washed 5X with 300 µL TBS-T, followed by addition of substrate,1.0 mg/ml pNPP (Sigma, N2770). Plates were incubated in the dark at room temperature for ~20 minutes before the addition of 1M Sodium hydroxide to stop the reaction. Absorbance was read at 405 nm on an M5 SpectraMax (Molecular devices). For data analysis, the blank OD units were subtracted from all wells, and the OD units for each calibrator were plotted against the calibrator concentrations and fit with a 4-parameter logistical fit. The concentrations of each replicate of unknowns and the back-calculated concentrations of the standards were interpolated to determine the ng/mL of TTR.
Quantitation of siRNA
Quantitation of siRNAs was performed by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS), similar to the methods described previously49,50. Briefly, tissue samples were homogenized, and tissue homogenates were processed by solid phase extraction using a Clarity OTX 96-well plate (Phenomenex) according to the manufacturer’s instructions, and the extracted samples were analyzed by LC-HRMS. The mobile phases used were as follows: mobile phase A: H2O/HFIP/DIEA (100:1:0.1, v/v/v) with 10 µM EDTA, mobile phase B: H2O/ACN/HFIP/DIEA (35:65:0.75:0.0375, v/v/v/v) with 10 µM EDTA. The column used was DNAPac RP column (4 µm, 50 x 2.1 mm; Thermo Fisher). Column temperature was set at 90 °C and flow rate was 0.2-0.3 mL/minute. The gradient started with 10% B, progressed to 50% B over 2.5 minutes, and then increased to 100% B in 0.2 minute and maintained for 0.8 minutes; the column was then re-equilibrated with 10% B for 1.5 minutes. A Dionex UltiMate 3000 HPLC system (Thermo Fisher) in combination with an Accela Open Autosampler (Thermo Fisher) and a Q Exactive mass spectrometer (Thermo Fisher) was used for the LC-HRMS analysis. The oligonucleotides were analyzed in negative ionization mode. The mass spectrometer was set at parallel reaction monitoring mode.
Histopathology, IHC, and ISH
All animals were sacrificed in accordance with AVMA Guidelines for the Euthanasia of Animals. Tissues were collected51 and fixed in 10% neutral-buffered formalin, processed routinely, and stained with hematoxylin and eosin (H&E) as described previously10. H&E based microscopic findings were recorded in the Pristima v7.0.0 System (Xybion, NJ) and graded on a scale of 1-5, (1=minimal, 2= mild, 3=moderate, 4=marked, and 5=severe) by a board-certified veterinary pathologist.
Luxol fast blue/cresyl-echt violet (LFB/CEV) stain for myelin was performed on spinal cord, brainstem and cerebellum. Briefly, deparaffinized sections were dehydrated through three changes in xylene, 2 minutes each, and rehydrated through two changes of 100% and 95% ethanol, 2 minutes each. Sections were stained with 0.1 % LFB (StatLab, Columbia, MD) either for 1 hour at 60° C or overnight at room temperature, followed by three to five rinses in 95% ethanol, distilled water, and differentiation in 0.05% lithium carbonate for three to five seconds. Next, sections were rinsed in distilled water followed by counter staining with CEV for 10 minutes, dehydrated through graded alcohols and xylene, and routinely coverslipped. Decreases in LFB staining intensity and increases in IBA1 staining indicative of microgliosis were semi-quantitatively scored on a scale of 1-4 (1=minimal, 2= mild, 3=moderate, and 4=marked).
Immunohistochemistry (IHC) and in-situ hybridization (ISH) were performed on the Discovery Ultra automated instrument (Ventana Medical Systems, Tucson, AZ) using manufacturer-provided reagents and protocols. For IHC, antibodies against the following targets were used: IBA1 1:1600 (#019-19741, Fujifilm Wako, Richmond, VA), GFAP 1:4000 (#Z0334, Agilent-Dako, Santa Clara, CA), CD31 1:75 (#ab23874, Abcam, Waltham, MA), MAP2 1:1000 (#ab5392, Abcam), TTR 1:800 (prealbumin, #SC-8104, Santa Cruz Biotechnology, Dallas, TX), and in-house anti-siRNA rabbit polyclonal antibody ab19151 at 1:12000. IBA1, GFAP, and CD31 were detected with anti-rabbit NP multimer followed by anti-NP AP multimer and Discovery Yellow AP Chromagen (all Ventana). MAP2 was detected with goat anti-chicken antibody (#6100-08, Southern Biotech, Birmingham, AL), followed by anti-goat HRP secondary antibody and Discovery Green HRP Chromogen (both Ventana). TTR was detected by anti-goat HRP secondary antibody followed by Discovery Purple HRP Chromogen (both Ventana). siRNA was detected with anti-rabbit HRP secondary antibody followed by Discovery Purple HRP Chromogen (Ventana). For ISH, sections were hybridized with a SOD1 mRNA probe (Advanced Cell Diagnostics, 428589) followed by detection with RNAScope DAB amplification kit (Advanced Cell Diagnostics, 323200) and visualization with mRNA DAB Chromogen (Ventana).