Cellular Analysis of Silencing the Huntington’s Disease Gene Using AAV9 Mediated Delivery of Artificial Micro RNA into the Striatum of Q140/Q140 Mice

Abstract

Background: The genetic mutation in Huntington’s disease (HD) is a CAG repeat expansion in the coding region of the huntingtin (Htt) gene. RNAi strategies have proven effective in substantially down-regulating Htt mRNA in the striatum through delivery of siRNAs or viral vectors based on whole tissue assays, but the extent of htt mRNA lowering in individual neurons is unknown.

Objective: Here we characterize the effect of an AAV9-GFP-miR^Htt vector on Htt mRNA levels in striatal neurons of Q140/Q140 knock-in mice.

Methods: HD mice received bilateral striatal injections of AAV9-GFP-miR^Htt or AAV9-GFP at 6 or 12 weeks and striata were evaluated at 6 months of age for levels of Htt mRNA and protein and for mRNA signal within striatal neurons using RNAscope multiplex fluorescence in situ hybridization.

Results: Compared to controls, the striatum of 6-month old mice treated at 6 or 12 weeks of age with AAV9-GFP-miR^Htt showed a reduction of 40–50% in Htt mRNA and lowering of 25–40% in protein levels. The number of Htt mRNA foci in medium spiny neurons (MSNs) of untreated Q140/Q140 mice varied widely per cell (0 to 34 per cell), with ∼10% of MSNs devoid of foci. AAV9-GFP-miR^Htt treatment shifted the distribution toward lower numbers and the percentage of cells without foci increased to 14–20%. The average number of Htt mRNA foci per MSN was reduced by 43%.

Conclusions: The findings here show that intrastriatal infusion of an AAV9-GFP-miR^Htt vector lowers mRNA expression of Htt in striatum by ∼50%, through a partial reduction in the number of copies of mutant Htt mRNAs per cell. These findings demonstrate at the neuronal level the variable levels of Htt mRNA expression in MSNs and the neuronal heterogeneity of RNAi dependent Htt mRNA knockdown.

Keywords

Huntington AAV microRNA RNAscope mRNA Htt huntingtin

INTRODUCTION

Huntington’s disease (HD) is an autosomal dominant disease caused by expansion of CAG repeats in exon 1 of the HTT gene leading to an increase in the polyglutamine repeat in the N-terminus of the Htt protein [1]. The mutant Htt protein accumulates in nuclear aggregates and causes a variety of changes that affect neuronal function [2]. Gene silencing is a promising mechanism to develop treatments for autosomal dominant diseases such as HD. A variety of gene silencing approaches based on antisense oligonucleotide (ASO), siRNAs, shRNAs, and microRNAs have been tested in HD models, each with advantages [3].

Adeno-associated virus (AAV) vectors encoding shRNA or artificial microRNAs (miR) have been used extensively for gene silencing studies in vivo due to exceptional gene transfer efficiency, broad tropism and stable transgene expression [4 –7]. Many studies have examined AAV-RNAi lowering of Htt in transgenic mouse models of Huntington’s disease. Striatal injection of an AAV1-encoded Htt-specific shRNA in transgenic N171-82Q mice reduced mRNA levels by ∼60% and resulted in improved behavioral performance [8]. AAV1-mediated long-term expression of an shRNA from a U6 promoter caused neuronal loss in the striatum of CAG140 knock-in mice; however, expression of an artificial miR from the same promoter was safe and effective in lowering Htt mRNA [9]. Additionally, intrastriatal injection of an AAV1 vector encoding an artificial miR under a pol II promoter in YAC128 mice reduced Htt mRNA and protein [10]. Vascular administration of an AAV9 vector encoding an artificial mRNA in BACHD and N171-82Q mice reduced Htt mRNA in the striatum by 25–35% [11]. Imaging analyses of striatal sections stained by immunohistochemistry indicated a 65% reduction in human Htt protein. The reason for this apparent discrepancy in silencing of Htt mRNA and protein is unclear. Despite extensive work in HD transgenic animals, few HD silencing studies have been carried out in knock-in mouse models. Compared to transgenic mice, knock-in models express full-length mutant Htt from the endogenous promoter. Previous studies have shown that by 6 months of age Q140/Q140 knock-in mice exhibit abnormalities in climbing, present neuronal inclusions, and have changes in levels of striatal enriched and synaptic proteins [12 –15].

The roughly 50% reduction in striatal Htt mRNA has been a consistent finding in studies investigating the therapeutic potential of AAV vectors encoding shRNA or artificial miRs by direct striatal injection or vascular delivery [8–11 , 16]. This partial silencing of Htt raises the question of whether only a subset of striatal cells are being transduced and are fully depleted of Htt mRNA or if AAV transduction is highly efficient, but there is only partial lowering of the Htt gene expression. Here we used in situ hybridization to assess Htt mRNA knock-down at the cellular level in the striatum of animals treated with AAV9-GFP-miR^Htt.

METHODS

Animals

Homozygous Q140/Q140 knock-in mice [12] on a mixed 129Sv/C57/BL6 background were bred and housed in the University of Massachusetts Medical School Animal Facility. The mice have human Htt exon 1 inserted into the mouse Htt gene. The Q140/Q140 mice breeding colony was supplemented by additional Q140/Q140 mice provided from Jackson Laboratories (Bar Harbor, ME). Mice were maintained on a 12 hour on and 12 hour off light schedule with unlimited access to food and water. Animals were kept in conventional mouse cages with filter tops. Genotypes were verified by PCR from DNA extracted from tail snips. The Institutional Animal Care and Use Committee at the University of Massachusetts Medical School reviewed and approved all animal experiments in compliance with the guidelines set forth by the NIH’s Guide for the Care and Use of LaboratoryAnimals.

Surgery

The Q140/Q140 mice were either 6 weeks or 12 weeks old at the time of surgery; After surgery mice were housed with littermates. Mice received bilateral injection of AAV9 vectors or PBS into the striatum at the following stereotaxic coordinates in relation to bregma (in mm): AP+1.0, ML±2.0, DV- 3.0 (from brain surface). The infusion rate was 0.125 μl/minute for a total volume of 2.17 μl for an AAV9 vector dose of 1×10¹⁰vg. Q140/Q140 mice were sacrificed at 26 weeks of age(6 months).

Constructs and viral vector production

We engineered a self-complementary AAV9-GFP-miR^Htt vector carrying a CBA promoter driven GFP expression cassette with two copies in tandem of an artificial microRNA against Htt embedded in the 3^′ untranslated region (3^′UTR) of GFP (Supplementary Figure 1). The sequences targeting mouse Htt mRNA were embedded in a miRNA miR-155 (mir^Htt) scaf-fold 5^′-ctggaggcttgctgaaggctgtatgctgTTTAGACTTGTGTCCTTGACCTgttttggccactgactgacTGGCAAAGCACAAGTCTAAAcaggacacaaggcctgttactagcactcacatggaacaaatggcc- 3^′ (guide strand is shown in bold). miR^Htt targets position 1090 in Exon8 of the mouse Htt gene. The transgene cassette composed of the cDNA encoding green fluorescent protein (GFP) followed by two tandem copies of miR^Htt in the 3^′UTR was expressed under the control of the chimeric cytomegalovirus enhancer/chicken β-actin promoter (CBA) followed by a chimeric intron [17] (Supplementary Figure 1). AAV9 vectors were produced, purified and titers were determined as previously described [18].

Tissue collection

Brains were harvested from mice and placed in ice-cold artificial cerebral spinal fluid (CSF). The cerebellum was removed before mounting the remaining brain on the vibratome platform. A block of 3% agarose was glued to the posterior part of the brain to steady it for cutting. 300 μm slices spanning the striatum were prepared and 2 mm punch biopsies from three to four sections of the striatum per mouse (Miltex, Plainsboro New Jersey) were taken of the GFP fluorescence area in the striatum (visualized on a Nikon Eclipse TS100 inverted microscope). Brains and slices were kept cold in artificial CSF at all times. Punch biopsies were immediately frozen on dry ice and stored at–80°C.

Determination of RNA levels by qPCR

Total RNA was isolated from tissue punches using a motorized hand homogenizer in 200 μl of Trizol (Life Technologies, Grand Island NY) according to manufacturers instructions. RNA was reverse transcribed using the High Capacity RNA to cDNA Kit (Applied Biosystems, Foster City CA). The following Taqman PCR probes (Applied Biosystems) for mouse genes were used in these studies: Htt (Assay ID: Mm01213820_m1), dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP32) (Assay ID: Mm00454892_m1), hypoxanthine phosphoribosyltransferase 1 (HPRT 1) (Assay ID: mm00446968_m1). Fold changes in htt mRNA expression levels across groups were determined using the ΔΔCt method. DARPP32 mRNA levels were compared across groups using ΔCt values, Hprt1 was used as a reference gene to normalize Ct values of Htt and DARPP32, and to calculate ΔCt values.

RNAscope in situ hybridization for GFP, Htt and DARPP32 mRNA

Whole brains were embedded in OCT compound (Tissue-Tek) and frozen in a 2-methylbutane dry ice bath. Ten μm thick sections were prepared using a cryostat and mounted on glass slides (SuperFrost, Fisher Scientific, Pittsburgh PA). Hybridization was performed using a RNAscope Multiplex Fluorescent kit (Advanced Cell Diagnostics, Hayward CA). Slide mounted sections were fixed in 10% phosphate buffered formalin at 4° for 15 min. Sections were dehydrated in increasing percentages of ethanol and incubated in Pretreat 4 solution for 30 minutes at room temperature. Hybridization with branched DNA probes specific for mRNA of GFP (400281), mouse Htt (405881), and DARPP32 (405901) was performed for 2 hr at 40°C in a hybridization oven (HybEZ Oven, Advanced Cell Diagnostics). Four amplification steps were carried out in the hybridization oven at 40°C with alternating incubations of 30 and 15 minutes. Samples were treated with DAPI for 30 seconds, then rinsed with PBS before applying a coverslip with ProLong Gold anti-fade (Life technologies). Images were taken using a LeicaDM5500B fluorescence microscope with a Leica DFC 365 Fx camera. Ten images per section and three sections per brain (injection, anterior and posterior to the injection) were taken using a 63x oil objective. A macro for the NIH Image J software was used to quantify the number of htt mRNA foci in DARPP32 positive and GFP positive cells. The outline of a neuron labeled for DARPP32 mRNA was defined and all Htt foci were counted within that region. The percentage of cells containing 0-2, 3-4 and 5 or more foci was compared using generalized linear models and the logic link to account for the constrained distribution of proportions. These models and between group contrasts were fit with the statistical software package STATA v13(StataCorp).

SDS-PAGE, western blot analysis and densitometry

Striatal tissue punches from both hemispheres were pooled into 1 tube and homogenized in a Dounce tissue grinder for (9 strokes) in 1.5 mL of solution composed of 0.32 M sucrose 10 mM DTT and protease inhibitor tablet (mini, cOmplete, EDTA-free, Roche, Indianapolis, IN). Protein concentrations were determined based on the Bradford method using Bio-Rad protein assay dye reagent. Homogenates were stored at –80°C until SDS-PAGE and western blot analysis. 3 μg of each crude homogenate sample was separated by SDS-PAGE using 3–8% Tris-acetate gels or 4–12% Bis-Tris gels (Life Technologies). Gels were transferred to nitrocellulose (Bio-Rad, Hercules CA) using TransBlot Turbo (Bio-Rad) and blots were cut into horizontal strips and blocked in 5% milk (Bio-Rad) in Tris buffered saline + 0.1% Tween20 (TBST) for 30 minutes at room temperature. Blots were incubated in primary antibody diluted in 5% milk/TBST overnight at 4°C with agitation and then washed in TBST. Blots were incubated in peroxidase labeled secondary antibodies diluted in 5% milk/TBST for 1 hour at room temperature, washed, then incubated in SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford IL) for 5 minutes and exposed to film (Hyperfilm ECL, GE Healthcare, Bukinghamshire, UK). Antibodies used for western blot analysis are as follows: Htt (rabbit polyclonal, Ab1, 1 : 2000 [19]), PDE10 (rabbit monoclonal, Abcam, 1 : 2000), DARPP32 (rabbit monoclonal, Abcam, 1 : 2000), GFP (rabbit monoclonal, Cell Signaling, 1 : 3000), GAPDH (mouse monoclonal, Millipore, 1 : 6000). Films were scanned with a flat bed scanner and densitometry was performed using NIH Image J software; the area of each band and its average intensity was measured to determine total intensity. GAPDH was used as a loading control and all signals were divided by the corresponding GAPDH signal for that animal as normalization. For each protein, the mean average intensity for each group was determined and an unpaired Student’s t-test was performed. The signal of each AAV9-GFP-miR^Htt animal was divided by the mean control group signal (AAV9-GFP) to determine percent of control and bar graphs represent normalized signal intensity as a percent of mean control group signal ± standard deviation (SD).

Perfusions, brain sectioning and immunoperoxidase method

Mice were anesthetized with an overdose of tribromoethanol and perfused with phosphate buffered saline (PBS), followed by 4% paraformaldehyde in 1x PBS. Brains were removed and post-fixed in 4% paraformaldehyde for 4-5 hr at 4°C. Brain sections through the striatum (40 μm thick) were prepared using a Vibratome s1000 (Leica Biosystems). A series containing every sixth section through the rostro-caudal extent of the striatum was used for immunoperoxidase labeling with each primary antibody. The blocking steps were as follows: the first blocking step was 3% hydrogen peroxide for 3 min followed by two washes in PBS for 5 min each. Next was a 20 min incubation in 0.5% triton x-100 (Sigma T8787) followed by two 5 min washes in PBS. Finally the sections were blocked in 2.5% horse serum for one hour. A series of sections was incubated with primary antibodies diluted in blocking buffer overnight at 4°C, followed by detection with an immunoperoxidase kit: anti-DARPP32 rabbit monoclonal (Abcam # ab40801) concentration-1 : 50,000, anti-Iba1 (Wako 019-19741) concentration- 1 : 1,000. This was followed by ImmPRESS Anti-Mouse Ig kit(Vector Laboratories # MP-7402). Controls consisted of omission of the primary antibody. For detection of the diaminobenzidine (DAB) reaction product, we used the Metal Enhanced DAB kit (Thermo Fisher Scientific # PI34065). All sections were mounted on slides coated with 0.5% gelatin (Sigma G-2500) and allowed to dry overnight. They were submerged in xylenes for 2 minutes and coverslips were applied using Cytoseal 60 (Richard-AllenScientific).

Measurement and quantification of DARPP32 labeling in the striatum

After DARPP32 immunostaining, digital images of tissue sections were generated using a flatbed scanner and DARPP32-positive stained striatum was manually identified in each section. Cross sectional area was measured using NIH ImageJ software.

RESULTS

Analysis of Htt silencing in Q140/Q140

We used a self-complementary AAV9-GFP-miR^Htt vector carrying a CBA promoter driven GFP expression cassette with two copies in tandem of an artificial microRNA against mouse Htt (miR^Htt) embedded in the 3^′UTR (Supplementary Figure 1). We injected AAV9-GFP-miR^Htt, or AAV9-GFP bilaterally into the striatum of Q140/Q140 mice at either 6 weeks or 12 weeks of age and collected striatal samples at 6 months of age. In mice injected with AAV9-GFP-miR^Htt, the striatal levels of mutant Htt (mHtt) mRNA were 40–50% lower than in control mice injected with AAV9-GFP (Fig. 1A), while DARPP32 mRNA remained unchanged (Fig. 1B). Western blot showed that mutant Htt protein was reduced in mice treated with AAV9-GFP-miR^Htt vector at 6 and 12 weeks of age by 40% and 25% (p < 0.05) respectively, compared to mice treated with AAV9-GFP (Fig. 2A-C). In the 6-week group the variability in Htt expression levels as represented by the relatively large standard deviations could be related to limiting amount of total protein isolated from some striatal punches. We examined protein levels of DARPP32 and PDE10, which are enriched in the striatum and are localized to medium spiny neurons and found no differences between groups (Fig. 2A,C,D).

Neuronal analysis of mutant Htt mRNA silencing in Q140/Q140 mice by in situ hybridization.

Our results showed that AAV9-GFP-miR^Htt reduced mutant Htt mRNA in striatum. To further investigate reduction of striatal Htt at the cellular level, we used a branched DNA-based in situ hybridization method (RNAscope) to detect and quantify changes in Htt mRNA levels in MSNs (DARPP32-positive cells) in the striatum (Fig. 3). Using this approach target mRNAs can be amplified 20x revealing punctate staining thought to represent single RNA molecules. Htt, DARPP32 and GFP mRNA foci were analyzed in striatal sections of Q140/Q140 mice injected at 6 weeks of age with AAV9-GFP-miR^Htt, AAV9-GFP or non-injected mice (Fig. 3A). The number of Htt mRNA foci in MSNs of control groups (AAV9-GFP and non-injected) varied between 0 and 34 per cell, with 2–5 Htt mRNA foci per MSN occurring in ∼56% of MSNs analyzed regardless of location in the striatum. A marked shift in the distribution of Htt mRNA foci in MSNs of mice injected with AAV9-GFP-miR^Htt was apparent; the percent of total MSNs with 0-2 foci was higher in the AAV9-GFP-miR^Htt group compared to control groups (p < 0.05; Fig. 3B). Conversely the percentage of MSNs with ≥5 foci was lower in AAV9-GFP-miR^Htt compared to controls (p < 0.05; Fig. 3D). These results were statistically significant at the injection site as well as in striatal regions caudal and rostral to the injection site. Taken together, the in situ hybridization results indicate that AAV9-GFP-miR^Htt lowers the levels of mutant Htt mRNA in the majority of transduced MSNs. In control groups, the percentage of MSNs devoid of Htt mRNA foci was 9-10%. Treatment of Q140/Q140 mice with AAV9-GFP-miR^Htt increased the percentage of cells with no Htt mRNA foci to 14–20% of MSNs, depending on the striatum region analyzed (Supplementary Figure 2). The average number of Htt mRNA foci per cell was 2.6±0.46 in the AAV9-GFP-miR^Htt group compared to 4.6±0.24 in the AAV9-GFP group (mean±standard deviation; p < 0.0002; Student’s t-test), which corresponds to a 43% reduction in Htt mRNA foci.

Impact of AAV9-GFP-miR^Htt in striatum of Q140/Q140 mice

AAV9-GFP-miR^Htt silencing of Htt in the Q140/Q140 mouse striatum appeared to be well tolerated as the intensity of DARPP32 staining was comparable across AAV9 injected groups and non-injected controls (Fig. 4A). This suggests that neither AAV vector injection nor miR^Htt expression induce measurable loss of MSNs. Also we found no difference in Iba1 immunoreactivity in the striatum of AAV9-injected mice compared to non-injected controls, suggesting the absence of an inflammatory response (Fig. 4B). The mean striatal cross sectional area of the striatum in most AAV9-injected mice was identical to that in non-injected controls, except in mice injected with AAV9-GFP-miR^Htt at 12 weeks of age where there was a 10% reduction(Fig. 4C).

DISCUSSION

In this study of HD Q140/Q140 mice, we achieved ∼40–50% reduction in mutant htt mRNA and 25–40% reduction in mutant Htt protein levels in the striatum using treatment with AAV9-GFP-miR^Htt. A maximum of 50% reduction in Htt mRNA expression in mouse striatum is a consistent finding in publications on AAV-mediated RNAi gene therapy for HD [8 , 16]. The reason for this apparent limit in Htt silencing in striatum is not understood. Generally, Htt gene silencing is measured by quantitative RT-PCR, but this method does not provide information at the single cell level. By using RNAscope in situ hybridization we found that AAV9-GFP-miR^Htt treatment caused a shift in the distribution of Htt mRNA in MSNs, implying not only reduced expression of mutant Htt protein, but also that treated cells are likely to retain some low level Htt expression. A major concern with the therapeutic reduction of Htt has been that complete elimination of Htt mRNA in individual neurons could be harmful, since Htt is a critical protein for normal neuronal physiology. However, a recent study has shown that genetic deletion of Htt in adult neurons does not cause neurodegeneration, or behavioral alterations [20]. Consistent with this report, and previous studies using AAV encoded miRs to silence Htt [8 , 16], we did not observe any indication of toxicity associated with AAV9-GFP-mir^Htt treatment despite documenting an increase in the number of cells devoid of Htt mRNA signal. There was no change in DARPP32 mRNA, protein levels or the immunoreactivity for DARPP32 in the striatum. Moreover the absence of an inflammatory response as reflected by no changes in Iba1 immunoreactivity is a further indication that the degree of Htt lowering documented here was well tolerated.

We found a wide range of Htt mRNA foci in DARPP32 positive MSNs varying from 0 to 34 foci per cell. Since Q140/Q140 mice express the poly Q expanded Htt from the endogenous promoter, it is possible that these results implicate biologicallyrelevant variability in Htt mRNA expression in MSNs. Further studies will be necessary to determine the significance of the neuronal variability in mutant Htt mRNA levels, and whether it identifies subsets of MSNs. Single-cell RNA sequencing data suggests the amount of mRNA for a specific gene can vary from cell to cell in specific cell types [21]. Moreover it is also possible that the snapshot of Htt mRNA expression in MSNs afforded by in situ hybridization represents the temporal regulation of gene expression in a population where each cell likely responds to a particular signaling environment that drives changes in gene expression over time.

In this study, we only evaluated the contribution of Htt mRNA reduction in MSNs to the overall effect of AAV9-GFP-miR^Htt in the striatum, and did not account for other cell types resident in the striatum, such as microglia and astrocytes. Although we stained sections with probes for microglia and astrocyte enriched genes such as Itgam (CD11b) and Alhd1 (data not shown), respectively, but the low number of foci and small cell bodies made it impossible to reliably apply the same counting strategy used for MSNs in those cell populations. Despite this limitation in our study it is important to consider that the average number of Htt mRNA foci per MSN transduced with AAV9-GFP-miR^Htt was 43% lower than for AAV9-GFP, and this change is in the same range as the ∼50% reduction in Htt mRNA levels quantified by RT-qPCR. The comparable change in Htt mRNA levels in GFP positive striatal regions assessed by two different methods suggests the contribution from those cell populations to the total Htt mRNA content in the striatum may be relatively small. However a study using conditional knockout HD mice showed that >50% reduction of Htt in striatum was achievable only by deleting the Htt allele simultaneously in cortex and striatum [22]. This suggests that corticostriatal axons from pyramidal neurons in layer V may contain Htt protein and possibly mRNA. However Htt silencing in those cortical neurons is unlikely to be effectively targeted in mice after striatal injection of AAV vectors encoding Htt-specific miRs. Although retrograde transport of AAV vectors has been documented in mice [23, 24], the efficiency of this process in the corticostriatal pathway after striatal injections in mice appears to be minimal compared to that documented in rats [25] and non-human primates [26, 27].

In summary, AAV9-GFP-miR^Htt treatment substantially reduced expression of both mHtt mRNA and protein and was well tolerated. In situ hybridization revealed a large variability in the content of mutant Htt mRNA in MSNs, and AAV9-GFP-miR^Htt treatment caused an overall shift toward lower content in the distribution of Htt mRNA in the majority of MSNs and eliminated it or reduced it below the detection limit of RNAscope in a small subset of these neurons. Our findings suggest that AAV mediated delivery of a non-allele specific artificial miR for Huntington’s disease was well tolerated as it does not appear to eliminate Htt expression completely, and reduced Htt levels may be sufficient to slow disease progression significantly.

AUTHOR DISCLOSURE STATEMENT

The authors have nothing to disclose.

Footnotes

ACKNOWLEDGMENTS

Eric Mick for statistical analysis. Funding sources: CHDI, Inc (NA, MSE, MD), and NIH NS38194 (NA, MD).

Appendix

References

A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Grou. Cell. 1993;72(6):971–83.

DiFiglia

, Sapp

, Chase

, Davies

, Bates

, Vonsattel

, et al. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science1997;277(5334):1990–3.

Aronin

, DiFiglia

. Huntingtin-lowering strategies in Huntington’s disease: Antisense oligonucleotides, small RNAs, and gene editing. Mov Disord. 2014;29(11):1455–61.

Miyazaki

, Adachi

, Katsuno

, Minamiyama

, Jiang

, Huang

, et al. Viral delivery of miR-196a ameliorates the SBMA phenotype via the silencing of CELF2. Nat Med. 2012;18(7):1136–41.

Kota

, Chivukula

, O’Donnell

, Wentzel

, Montgomery

, Hwang

, et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell. 2009;137(6):1005–17.

Stoica

, Todeasa

, Cabrera

, Salameh

, ElMallah

, Mueller

, et al. Adeno-associated virus-delivered artificial microRNA extends survival and delays paralysis in an amyotrophic lateral sclerosis mouse model. Ann Neurol. 2016;79(4):687–700.

Xia

, Mao

, Eliason

, Harper

, Martins

, Orr

, et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med. 2004;10(8):816–20.

Harper

, Staber

, He

, Eliason

, Martins

, Mao

, et al. RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proc Natl Acad Sci U S A.. 2005;102(16):5820–5.

McBride

, Boudreau

, Harper

, Staber

, Monteys

, Martins

, et al. Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi. Proc Natl Acad Sci U S A. 2008;105(15):5868–73 .

10.

Stanek

, Sardi

, Mastis

, Richards

, Treleaven

, Taksir

, et al. Silencing mutant huntingtin by adeno-associated virus-mediated RNA interference ameliorates disease manifestations in the YAC128 mouse model of Huntington’s disease. Hum Gene Ther. 2014;25(5):461–74.

11.

Dufour

, Smith

, Clark

, Walker

, McBride

. Intrajugular vein delivery of AAV9-RNAi prevents neuropathological changes and weight loss in Huntington’s disease mice. Mol Ther. 2014;22(4):797–810.

12.

Menalled

, Sison

, Dragatsis

, Zeitlin

, Chesselet

. Time course of early motor and neuropathological anomalies in a knock-in mouse model of Huntington’s disease with 140 CAG repeats. J Comp Neurol. 2003;465(1):11–26.

13.

Hickey

, Kosmalska

, Enayati

, Cohen

, Zeitlin

, Levine

, et al. Extensive early motor and non-motor behavioral deficits are followed by striatal neuronal loss in knock-in Huntington’s disease mice. Neuroscience. 2008;157(1):280–95.

14.

Valencia

, Sapp

, Kimm

, McClory

, Ansong

, Yohrling

, et al. Striatal synaptosomes from Hdh140Q/140Q knock-in mice have altered protein levels, novel sites of methionine oxidation, and excess glutamate release after stimulation. J Huntingtons Dis. 2013;2(4):459–75.

15.

Franich

, Fitzsimons

, Fong

, Klugmann

, During

, Young

. AAV vector-mediated RNAi of mutant huntingtin expression is neuroprotective in a novel genetic rat model of Huntington’s disease. Mol Ther. 2008;16(5):947–56.

16.

Boudreau

, McBride

, Martins

, Shen

, Xing

, Carter

, et al. Nonallele-specific silencing of mutant and wild-type huntingtin demonstrates therapeutic efficacy in Huntington’s disease mice. Mol Ther. 2009;17(6):1053–63.

17.

Mueller

, Tang

, Gruntman

, Blomenkamp

, Teckman

, Song

, et al. Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles. Mol Ther. 2012;20(3):590–600.

18.

Gao

, Alvira

, Somanathan

, Lu

, Vandenberghe

, Rux

, et al. Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc Natl Acad Sci U S A. 2003;100(10):6081–6.

19.

DiFiglia

, Sapp

, Chase

, Schwarz

, Meloni

, Young

, et al. Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron. 1995;14(5):1075–81.

20.

Wang

, Liu

, Gaertig

, Li

. Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis. Proc Natl Acad Sci U S A. 2016;113(12):3359–64.

21.

Dueck

, Khaladkar

, Kim

, Spaethling

, Francis

, Suresh

, et al. Deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation. Genome Biol. 2015;16:122.

22.

Wang

, Gray

, Lu

, Cantle

, Holley

, Greiner

, et al. Neuronal targets for reducing mutant huntingtin expression to ameliorate disease in a mouse model of Huntington’s disease. Nat Med. 2014;20(5):536–41.

23.

Cearley

, Wolfe

. Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain. Mol Ther.. 2006;13(3):528–37.

24.

Cearley

, Wolfe

. A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease. J Neurosci. 2007;27(37):9928–40.

25.

Salegio

, Samaranch

, Kells

, Mittermeyer

, San Sebastian

, Zhou

, et al. Axonal transport of adeno-associated viral vectors is serotype-dependent. Gene Ther. 2013;20(3):348–52.

26.

San Sebastian

, Samaranch

, Heller

, Kells

, Bringas

, Pivirotto

, et al. Adeno-associated virus type 6 is retrogradely transported in the non-human primate brain. Gene Ther. 2013;20(12):1178–83.

27.

Hadaczek

, Stanek

, Ciesielska

, Sudhakar

, Samaranch

, Pivirotto

, et al. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: Implications for Huntington’s disease. Mol Ther Methods Clin Dev. 2016;3:16037.

Cellular Analysis of Silencing the Huntington’s Disease Gene Using AAV9 Mediated Delivery of Artificial Micro RNA into the Striatum of Q140/Q140 Mice

Abstract

Keywords

INTRODUCTION

METHODS

Animals

Surgery

Constructs and viral vector production

Tissue collection

Determination of RNA levels by qPCR

RNAscope in situ hybridization for GFP, Htt and DARPP32 mRNA

SDS-PAGE, western blot analysis and densitometry

Perfusions, brain sectioning and immunoperoxidase method

Measurement and quantification of DARPP32 labeling in the striatum

RESULTS

Analysis of Htt silencing in Q140/Q140

Neuronal analysis of mutant Htt mRNA silencing in Q140/Q140 mice by in situ hybridization.

Impact of AAV9-GFP-miRHtt in striatum of Q140/Q140 mice

DISCUSSION

AUTHOR DISCLOSURE STATEMENT

Footnotes

ACKNOWLEDGMENTS

Appendix

References

Impact of AAV9-GFP-miR^Htt in striatum of Q140/Q140 mice