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. 2015 Dec 1;4(12):e266.
doi: 10.1038/mtna.2015.38.

Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

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Free PMC article

Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

Julia F Alterman et al. Mol Ther Nucleic Acids. .
Free PMC article

Abstract

Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

Figures

Figure 1
Figure 1
hsiRNAs are efficiently internalized by primary cortical neurons. (a) Schematic structure of hsiRNAs. A double-stranded oligonucleotide with single-stranded, phosphorothioated tale. 2′-O-methyl and 2′-fluoro modifications, conjugated to teg-chol. (b) Fluorescent images of primary cortical neurons incubated with 0.5 µmol/l Cy3-PPIB hsiRNA (red). Nuclei counterstained with Hoechst dye (blue), imaged on Zeiss confocal microscope, ×63. Bar = 10 µm. Images are representative, results confirmed in five separate experiments. (c) Primary cortical neurons incubated for 72 hours with hsiRNA targeting Ppib at concentrations shown. Level of Ppib mRNA was measured using QuantiGene (Affymetrix) normalized to housekeeping gene, Htt, presented as percent of untreated control (n = 3 wells, mean ± SD). NTC, nontargeting control (0.75 µmol/l). Graph is representative, results confirmed in three separate experiments.
Figure 2
Figure 2
Systematic screen identifies functional hsiRNAs targeting huntingtin mRNA. (a) Huntingtin mRNA levels in HeLa cells treated for 72 hours with 94 hsiRNAs (1.5 µmol/l) were quantified using QuantiGene and normalized to the housekeeping gene Ppib. Data are presented as percent of untreated control (n = 3 wells, mean ± SD). Gray area represents range of huntingtin mRNA levels encompassing untreated and nontargeting hsiRNA controls. Red bars indicate compounds selected for further analysis. Compound sequence, chemical composition, and level of silencing are shown in Supplementary Table S1. Graph is representative, results confirmed in two separate experiments. (b,c) Dose–response analysis of huntingtin mRNA levels in HeLa cells treated with HTT10150 hsiRNA (circles) or unmodified siRNA (squares) added to culture medium in the (b) absence (modified HTT10150 IC50 = 82.2 nmol/l) or (c) presence (modified HTT10150 IC50 = 0.004 nmol/l, unmodified HTT10150 IC50 = 0.013 nmol/l), of cationic lipids for 72 hours. Huntingtin mRNA was measured as described in a (n = 3 wells, mean ± SD). IC50 values were calculated as described in Materials and Methods and are presented in Supplementary Table S1. Graph is representative, results for modified siRNA confirmed in three separate experiments (in both absence and presence of cationic lipids), results for unmodified siRNA confirmed in two separate experiments (in both absence and presence of cationic lipids).
Figure 3
Figure 3
HTT10150 shows dose-dependent silencing of huntingtin by passive uptake in primary neurons. (a) Huntingtin mRNA levels in primary striatal (black) or cortical (gray) neurons 1 week after treatment with the indicated concentrations of HTT10150. Huntingtin mRNA levels were normalized to Ppib mRNA. Data are expressed as percent of untreated control (n = 3 wells, mean ± SD). NTC, nontargeting control (1.25 µmol/l). (b) Huntingtin protein levels in primary neurons 1 week after treatment with the indicated concentrations of HTT10150. Huntingtin and β-tubulin proteins were quantified by densitometry of western blots, and huntingtin protein levels were normalized to β-tubulin. Data are expressed relative to the level of huntingtin protein in untreated control cells. (n = 5 neuronal preparations from separate pups, mean ± SD). NTC, nontargeting control (1.25 µmol/l). Graph of silencing in primary cortical neurons after 1 week is representative, results confirmed in five separate experiments. (c) Original western blots from graph in b. Primary cortical neurons were cultured from five individual pups (#1–5) and incubated with HTT10150 at concentrations shown for 1 week. Huntingtin protein levels were detected by western blot using antibody AB1 (Huntingtin 1–17). NTC, nontargeting control. (d) Primary neurons were incubated with HTT10150 at concentrations shown, for 1, 2, and 3 weeks. Level of huntingtin mRNA was measured using QuantiGene (Affymetrix) normalized to housekeeping gene, Ppib (cyclophillin B), and presented as percent of untreated control (n = 3 wells, mean ± SD). NTC, nontargeting control (1.5 µmol/l). Graph of silencing in primary cortical neurons after 1 week is representative, results confirmed in five separate experiments. (e) Primary cortical neurons were incubated with two different Htt hsiRNA sequences HTT10150 and HTT10146 at concentrations shown for 72 hours. Level of huntingtin mRNA was measured using QuantiGene (Affymetrix) normalized to housekeeping gene, Ppib (cyclophillin B), and presented as percent of untreated control (n = 3 wells, mean ± SD). NTC, nontargeting control (1.5 µmol/l). Graph of HTT10150 silencing in primary cortical neurons after 72 hours is representative, results confirmed in seven separate experiments.
Figure 4
Figure 4
A single intrastriatal injection of HTT10150 is localized to neurons and fiber tracts ipsilateral to the injection site after 24 hours. Twenty-five micrograms of Cy3-HTT10150 (red) was unilaterally injected into the striatum of WT (FVB/NJ) mice. Brains were collected after 24 hours, paraffin embedded, and sectioned. (a) Tiled image of coronal brain section (×16). Majority of HTT10150 is localized at site of injection with sharp gradient of diffusion. (b) Tiled image of sagittal brain section (×16), injected side. (c) Image of coronal brain section (×40), injected side. (d) Image of coronal brain section (×60), injected side, with NeuN-stained neurons. (e) NeuN-stained neurons from injected side (×60) zoomed in. Solid arrow, NeuN staining. Open arrow, Cy3-HTT10150 punctae in perinuclear space. Images are representative, results confirmed in two separate experiments.
Figure 5
Figure 5
HTT10150 effectively silences huntingtin mRNA ipsilateral to the site of injection. HTT10150 was unilaterally injected into the striatum of WT (FVB/NJ) mice (2 µl). Mice were sacrificed at 5 days. Brains were sliced into 300-μm sections and six 2-mm punch biopsies of the (a) striatum and (b) cortex were collected from both ipsilateral and contralateral sides. Level of huntingtin mRNA was measured using QuantiGene (Affymetrix) normalized to housekeeping gene, Ppib (cyclophillin B), and presented as percent of untreated control (n = 8 mice, mean ± SD, three biopsies per region). P values are all calculated for each dose group relative to NTC by Two-way repeated-measures analysis of variance: 25 µg striatum, P < 0.0001; 12.5 µg striatum, P < 0.0001, P = 0.0002; 6.3 µg cortex, P = 0.0009. NTC, nontargeting control.
Figure 6
Figure 6
HTT10150 shows a twofold increase in microglial activation at the site of injection. HTT10150 was unilaterally injected into the striatum of WT (FVB/NJ) mice. Brains were collected after (b) 6 hours and (a, c) 5 days fixed, sectioned, and stained with antibodies against IBA-1. (a) Representative images of activated (black arrow) and resting (open arrow) after injection of 12.5 µg HTT10150 and ACSF 5 days post-injection, ×40 magnification. (b) Quantification of activated and resting microglia 6 hours post-injection of ACSF (n = 6 mice, mean ± SD) and 12.5 µg HTT10150 (n = 3 mice, mean ± SD). P values calculated by unpaired t-test, t = 9.996, df = 7: ACSF versus HTT10150 activated microglia ipsilateral striatum, P = 0.0239. ACSF versus HTT10150 activated microglia contralateral striatum, P < 0.0001. (c) Quantification of activated and resting microglia 5 days postinjection of ACSF (n = 4 mice, mean ± SD) and 12.5 µg HTT10150 (n = 3 mice, mean ± SD). Images are representative, results confirmed in separate images of all injected brains. P values calculated by unpaired t-test, t = 2.700, df = 5: ACSF versus HTT10150 activated microglia ipsilateral striatum P = 0.0428.
Figure 7
Figure 7
HTT10150 shows no toxicity in DARPP-32-positive neurons around the site of injection. HTT10150 was unilaterally injected into the striatum of WT (FVB/NJ) mice. Brains were collected after 5 days, fixed, sectioned, and stained with antibodies against DARPP-32 (a–d). Representative image of striatum after injection of (a,b) ACSF, full brain scan and ×60 magnification or (c,d) 12.5 µg HTT10150, full brain scan and ×60 magnification. (a) Quantification of DARPP-32–positive neurons (n = 3 mice, mean ± SD). Images are representative results confirmed in separate images of all injected brains.

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