spinocerebellar ataxia type 3 ( sca3 ) is one of nine polyglutamine expansion diseases and the most common dominantly inherited ataxia in the world .
while certain symptoms in sca3 may respond to symptomatic therapy , there is still no effective treatment for this relentlessly progressive and fatal neurodegenerative disease .
sca3 is caused by a cag repeat expansion in the atxn3 gene , resulting in an abnormally long polyglutamine stretch in the encoded atxn3 protein . because expression of mutant atxn3 is an early and necessary step in disease pathogenesis , strategies to reduce expression of the disease gene itself are high on the list of potential therapies.2 , 3 , 4 , 5 , 6 mice lacking the homologous atxn3 gene appear normal , suggesting that human atxn3 may not be an essential protein .
additionally , in a doxycycline - regulatable transgenic mouse model of sca3 , reducing production of mutant atxn3 transcripts via doxycycline treatment beginning at 9  weeks eliminated disease features .
this result implies that treatment at an early symptomatic stage , prior  to extensive irreversible tissue damage , holds promise for sca3 therapy . among gene suppression approaches ,
viral - mediated gene therapy has been explored by us and others in mouse models of sca3 . in transgenic mice expressing the full human atxn3 disease gene , bilateral delivery of small interfering rna targeting atxn3 to the deep cerebellar nuclei ( dcn )
was well tolerated and led to sustained suppression of mutant atxn3 in the cerebellum , but it did not rescue motor deficits and the lifespan in these mice . because degeneration in patients with sca3 extends beyond the cerebellum to include selective brainstem nuclei , the striatum , and the thalamus , effective gene therapy for sca3 may require broader cns delivery .
additional rnai studies targeting atxn3 succeeded in preventing motor deficits in a lentiviral mouse model of sca3 in which expanded atxn3 was virally delivered to the cerebellum,2 , 5 but these studies have not yet been replicated in mouse models expressing mutant atxn3 throughout the cns .
antisense oligonucleotides ( asos ) represent a therapeutic non - viral gene suppression approach that is increasingly being tested in neurodegenerative and metabolic disorders .
asos are short single strands of chemically modified oligonucleotides that selectively bind complementary mrna via watson - crick hybridization to drive rnase h - mediated cleavage of the targeted mrna .
asos targeting disease - causing genes have been tested in rodent models of various neurodegenerative disorders and have advanced into human clinical trials , including asos for spinal muscular atrophy ( sma ) , familial amyotrophic lateral sclerosis ( als ) , and huntington s disease ( hd).11 , 12 , 13 , 14 , 15 , 16 asos targeting atxn3 have been developed and tested in cellular and in  vivo models of sca3 , with some limitations.6 , 17 , 18 , 19 , 20 , 21 allele - specific asos that selectively target the cag repeat expansion in an effort to preserve wild - type atxn3 function have been tested in sca3 cellular models , but the ubiquity of cag repeats in the human transcriptome may pose challenging off - target effects.17 , 18 , 19 , 20 splice - switching asos have also been developed but failed to achieve greater than 50% knockdown of full - length mutant atxn3 in the aso - treated mouse cerebellum and did not preserve the deubiquitinating function of atxn3.6 , 21 here we evaluate the efficiency of non - allele - specific asos targeting human atxn3 in two complementary transgenic mouse models of sca3 : the yeast artificial chromosome ( yac ) mjd - q84.2 ( q84 ) model expressing the full - length human atxn3 disease gene with 84 cag repeats , and the cytomegalovirus ( cmv ) mjd - q135 ( q135 ) model expressing a single human atxn3 isoform from an atxn3 cdna with 135 cag repeats.22 , 23 overall , two candidate asos led to widespread delivery and efficient silencing of mutant human atxn3 with no immune response in the q84 mouse model of sca3 .
our results support the continued development of anti - atxn3 asos as therapy for sca3 .
to identify active atxn3 asos , asos complementary to human atxn3 coding and non - coding regions were screened at a single dose in hepg2 cells , a human cell line that expresses atxn3 transcripts .
following aso treatment , the ability of the asos to suppress normal atxn3 transcripts was quantified ( figure  1a ) .
the most active asos were subjected to dose response to determine their in  vitro median inhibitory concentration ( ic50 ) , and from these , five anti - atxn3 asos with similar ic50s ( 1.22  m ) ( figure  1b ) targeting coding or non - coding regions of the human atxn3 transcript were selected for further characterization ( figure  1c ) .
all five anti - atxn3 asos , denoted aso-1 through aso-5 , as well as a scrambled control aso ( aso - ctrl ) , shared a 5 - 8 - 5 2-o - methoxyethyl ( moe ) gapmer design with phosphorothioate backbone modifications , as well as 5-mer blocks of moe - modified ribonucleotides flanking an 8-mer block of deoxynucleotides without sugar modifications ( figure  1d ) .
this moe gapmer design has previously been shown to increase stability and potency of asos and is the design taken into clinical trials for als and hd.14 , 24 , 25 to assess target efficiency of asos in patient cells , sca3 patient - derived fibroblasts ( gm06153 ) were transfected with aso candidates , aso - ctrl , or pbs vehicle alone .
gm06153 fibroblasts are heterozygous for the mutant atxn3 allele harboring a cag expansion with 71 repeats and a non - pathological repeat of 23 .
cells were transfected with asos ( 4  m ) and harvested 48 or 72  hr later for rna or protein assessment , respectively .
all five asos targeting atxn3 robustly suppressed atxn3 transcript and protein levels ( p  < 0.0001 ) ( figures 1e and 1f ) .
only one of the five asos , aso-4 , exhibited any toxicity following transfection , with visible detachment of a fraction of treated cells ( data not shown ) .
all five aso candidates were subjected to in  vivo tolerability screening in non - transgenic mice .
animals received a single 700  g intracerebroventricular ( i.c.v . ) bolus injection into the right lateral ventricle , and animals were monitored for 7  weeks after injection .
aso - treated animals did not exhibit any overt behavioral deficits or differences in body weight compared to pbs - treated controls ( data not shown ) .
one aso-2-treated animal died during surgery , but this was deemed non - drug related . at the conclusion of the study , iba1 mrna in the cortex immediately adjacent to the injection site was quantified as a surrogate for microglial activation , and no significant changes were observed between aso groups and pbs ( data not shown ) .
thus , all five aso candidates were selected for further in  vivo assessment in the q84 transgenic mouse model of sca3 .
hemizygous q84 mice express the full - length human atxn3 gene with a pathogenic expansion of 84 cag repeats at near endogenous atxn3 expression levels , and thus represent a biologically relevant model of the human disease .
this mouse line has been validated in previous sca3 gene therapy studies.3 , 23 neuropathological features of sca3 , such as enhanced mutant atxn3 nuclear burden and the formation of cytoplasmic and intranuclear inclusions in neurons , are apparent as early as 6  weeks of age in hemizygous q84 mice.3 , 23 to assess whether anti - atxn3 aso intervention early in the disease process can suppress accumulation of mutant atxn3 protein in  vivo , we injected 8-week - old sex - matched hemizygous q84 mice with a single stereotaxic i.c.v .
each mouse received 500  g asos diluted in 10  l pbs at an injection rate of 0.5  l / s to enhance aso spread .
mice were subsequently monitored according to university of michigan committee on use and care of animals ( ucuca ) guidelines , with no post - surgical adverse events or deaths occurring .
the left hemisphere contralateral to injection was further dissected into the forebrain , cerebellum , diencephalon , and cervical spinal cord for protein and rna analysis , while the right hemisphere ipsilateral to the injection site was processed for immunohistochemical ( ihc ) analysis ( figure  2a ) .
all treatment groups were blinded to the experimenter prior to analysis . in sca3 , many neuronal populations in the diencephalon appear selectively vulnerable and exhibit atxn3-positive aggregate pathology.26 , 27 four of the five aso candidates significantly reduced mutant atxn3 transcripts in the diencephalon relative to vehicle - treated mice , assessed by qrt - pcr ( figure  2b ) . two asos targeting the 3-utr of atxn3 , aso-4 and aso-5 , achieved the greatest transcript reduction in the diencephalon , reducing mutant atxn3 transcript levels by 63.8%   6.8% ( p 
0.0001 ) , respectively , relative to vehicle - treated mice . aso-1- and aso-2-treated mice also showed significant transcript reduction in the diencephalon ( 58.2%   24.5% and 56.4%   23.7% , respectively ; p  < 0.01 )
. suppression of mutant atxn3 protein expression was assessed by western blot of left forebrain , cerebellum , diencephalon , and cervical spinal cord protein lysates from treated mice ( figures 2c and 2d ) . mirroring the reduction in diencephalon transcript levels , mutant atxn3 protein levels
were significantly reduced in the diencephalon , cerebellum , and cervical spinal cord of mice treated with aso-2 ( p  <  0.05 ) , aso-4 ( p  < 0.05 ) , or aso-5
( p  < 0.01 ) . additionally , a significant mutant atxn3 reduction was found in forebrain lysates of aso-2-treated ( p  < 0.05 ) and aso-5-treated ( p  <
in aso-1-treated mice , high variability in mutant atxn3 protein levels was observed in all analyzed brain regions , with four of six mice exhibiting mutant atxn3 suppression of more than 50% relative to vehicle - treated mice , while the remaining two mice exhibited no decrease in mutant atxn3 levels .
levels of high molecular weight ( hmw ) atxn3 aggregate species resolvable by sds - page were also quantified in diencephalon protein lysates from treated mice .
hmw atxn3 levels were significantly reduced by 63.7%   7.7% and 68.5   7.3% in the diencephalon of mice treated with aso-2 ( p  < 0.05 ) and aso-5 ( p  < 0.05 ) , respectively , relative to vehicle - treated q84 mice ( figures 2e and 2f ) .
hmw atxn3 trended toward a decrease in aso-4-treated mice , but failed to reach significance due to variability across mice ( p  = 0.09 ) .
analysis of diencephalon protein lysates also revealed a significant decrease in endogenous murine atxn3 protein in q84 mice treated with aso-1 ( 42.9%   8.3% ; p  < 0.05 ) , aso-2 ( 40.2%   14.4% ; p 
<  0.05 ) , aso-4 ( 42.0%   12.0% ; p  < 0.05 ) , and aso-5 ( 82.6%   9.3% ; p  <
0.0001 ) relative to vehicle - treated q84 mice ( figure  2 g ) .
of the five asos tested , only aso-5 was expected to directly target the murine mrna transcript due to species conservation of the target sequence between the human and murine atxn3 gene ( figure  2b ) .
previous studies have shown that mutant expanded atxn3 can oligomerize with non - expanded atxn3 , leading to increased retention of both pathogenic and non - pathogenic atxn3 proteins . thus ,
a reduction of murine atxn3 protein may reflect decreased interactions with the mutant protein leading to normalization of the protein clearance rate .
furthermore , vehicle - treated q84 mice exhibited paradoxical decreases in atxn3 transcript levels ( p  < 0.05 ) ( figure  2b ) and increased endogenous protein levels of murine atxn3 relative to vehicle - treated wild - type animals ( p  < 0.001 ) ( figure  2 g ) .
aso delivery and potency in disease - relevant neuronal populations were further investigated with anti - atxn3 and anti - aso immunofluorescence on brain sections from the injected hemisphere .
four  weeks following injection , all asos were widely distributed throughout the brain , including in the striatum , cerebellar lobules , dcn , pons , hippocampus , and cerebral cortex ( figure  3a ) .
areas of high vulnerability in sca3 , including the dcn and pontine nuclei , exhibited high levels of delivered aso and , correspondingly , clearly decreased levels of atxn3 expression in these regions ( figures 3b and 3c ) .
q84 mice treated with aso-2 , aso-4 , and aso-5 exhibited markedly decreased neuronal nuclear atxn3 staining in the dcn and pons ( p  < 0.0001 ) , two neuronal populations that are highly affected in sca3 ( figure  4 ) .
aso-5 led to the greatest reduction in atxn3 immunostaining in the dcn and pons , likely due to the fact that it is the only tested aso that targets both human and murine atxn3 transcripts . to investigate the tolerability and safety of asos in  vivo , we assessed transcript levels of markers of immune - reactive cell types  namely , astrocytes ( gfap ) and microglia ( iba1)in the diencephalon of treated mice .
gfap levels were not altered in mice treated with aso-2 or aso-5 but were significantly increased in mice treated with aso-4 ( p  < 0.05 ) ( figure  5a ) , the only aso that also exhibited cellular toxicity in patient fibroblasts .
iba1 transcript levels were not increased in the diencephalon of aso-2- or aso-4-treated mice , although iba1 levels trended toward an increase in aso-4-treated mice , mirroring the increased gfap transcript levels in aso-4-treated mice and further supporting the possible toxicity of this aso .
intriguingly , aso-5 treatment of q84 mice actually led to a significant decrease ( p  <
0.05 ) in iba1 transcript levels in the diencephalon relative to vehicle- or aso - ctrl - treated mice ( figure  5b ) . this result suggests a possible rescue of disease - related neuroinflammatory changes in sca3 mice by aso-5 treatment .
immunofluorescence assessment of reactive astrocytes and microglia was performed throughout the brain of the injected hemisphere .
unexpectedly , we noted a discrepancy in astrocyte - microglia patterning in the dcn of mice treated with aso-4 , specifically .
the dcn typically exhibits a pattern of gfap - positive astrocytes forming a rim around the nucleus , which in turn is dominated by iba1-positive microglia , with little overlap between the two cell types .
however , aso-4-treated mice consistently demonstrated infiltration of astrocytes from the surrounding region into the dcn , indicative of possible aso-4 toxicity leading to inflammatory changes in this brain region ( figure  5c ) .
all mice , including vehicle- , aso - ctrl- , and anti - atxn3 aso - treated mice , exhibited similar local gliosis immediately surrounding the injection site ( data not shown ) but , consistent with our earlier screens in wild - type mice , there were no differences in iba1 or gfap expression levels in the cerebral cortex neighboring the injection site , where local aso concentrations were high ( figure  5d ) .
no apparent difference in the density of gfap- and iba1-positive cells was identified in pontine nuclei or any other  assessed brain region of aso-2- and aso-5-treated mice .
qualitatively , however , more gfap - positive cells were apparent in the pons of aso-4-treated mice ( figure  5e ) .
the top aso candidates identified in q84 mice were similarly evaluated in hemizygous q135 mice , a transgenic model of sca3 that expresses an atxn3 cdna splice isoform with a 135-cag repeat expansion . of the three asos demonstrating significant atxn3 knockdown in brain regions of q84 mice , only aso-4 and aso-5 target sequences are present in the atxn3 cdna transcript expressed in q135 mice .
thus , only these two asos could be evaluated in q135 mice ( figure  6a ) .
bolus injection of 500  g aso-4 , 700  g aso-5 , 700  g aso - ctrl , or pbs into the right lateral ventricle , and brains were harvested 4  weeks later .
the aso-5 dose was increased to 700  g to increase potential efficacy , while the aso-4 dose was maintained at 500  g due to toxicity concerns from previous q84 mouse studies .
as in the q84 mouse experiment , q135 brains were bisected along the midline with the left hemisphere used for protein and rna analysis and the right hemisphere for immunofluorescence with anti - aso and anti - atxn3 antibodies .
mutant atxn3 transcript levels were not significantly reduced in the  diencephalon of either aso-4- or aso-5-treated mice relative to vehicle or aso - ctrl - treated mice , assessed by qrt - pcr ( figure  6b ) . in contrast , wild - type atxn3 transcript levels were significantly reduced ( p  < 0.0001 ) in aso-5-treated mice , but not in aso-4-treated mice , consistent with the conservation of target sequence for aso-5 but not aso-4 .
this reduction in wild - type atxn3 transcript levels confirms effective aso-5 delivery to the diencephalon for targeting but also supports a greatly reduced targeting capacity of the asos for  the cdna transgene relative to the wild - type gene .
immunoblotting for atxn3 also revealed no significant difference in mutant atxn3 levels in the cerebellum of treated mice ( figure  6c ) .
anti - aso immunofluorescence confirmed that asos were delivered successfully to the cerebellum and pons of q135 mice , as in q84 mice ( figures 6d and 6e ) .
anti - atxn3 fluorescence revealed no visible difference in diffuse atxn3 protein staining or in atxn3-positive punctate pathology in aso-4-treated mice relative to aso - ctrl- and vehicle - treated mice in the dcn ( figure  6d ) or pons ( figure  6e ) .
aso-5-treated mice exhibited almost total silencing of diffuse atxn3 staining , which we attribute to efficient knockdown of endogenous murine atxn3 .
however , atxn3-positive punctate neuropathology did not appear markedly reduced in the q135 pontine nuclei of 12-week - old mice ( figure  6e ) .
our studies here strongly support the promise of aso - mediated targeting of atxn3 as a potentially effective therapeutic approach for sca3 .
several tested asos were effective in reducing levels of the pathogenic atxn3 protein both in human disease fibroblasts and in a mouse model expressing the full - length human mutant atxn3 gene .
sca3 brains exhibit widespread degenerative  and neuropathological changes , including neuronal loss and gliosis in the dcn , vestibular and other brainstem nuclei , spinocerebellar tracts , substantia nigra , thalamus , and globus pallidus.26 , 29 this broad distribution highlights the need for broad targeting of a therapeutic agent to affected brain regions .
here we identified several asos that effectively target the full - length atxn3 human transcript in sca3 patient fibroblasts , including exonic , intronic , and 3-utr targets .
all five asos were carried forward in a short - term assessment in the q84 mouse model of sca3 .
similar to previous aso studies,16 , 30 , 31 , 32 we directly injected asos into the lateral ventricle to allow the natural flow of the cerebrospinal fluid to deliver the aso therapy to affected brain regions of interest .
similar broad aso delivery throughout the cns was shown in other rodent models following i.c.v .
importantly , for three of the five tested asos , a  single aso injection into symptomatic q84 transgenic mice was sufficient to significantly reduce ( > 50% ) atxn3 protein levels in the  cerebellum , diencephalon , and  cervical spinal cord .
the top aso candidates also caused a marked reduction in hmw aggregated atxn3 in the diencephalon , detectable on denaturing gels .
in contrast to the robust suppression in q84 mice , we failed to observe robust aso - mediated reduction of mutant atxn3 in a second transgenic model , the q135 mouse .
this lack of efficacy was not due to insufficient delivery , since aso-5 reduced endogenous atxn3 levels in q135 mice much as it did in q84 mice .
decreased potency of aso targeting in q135 mice may result from key differences between cdna - derived and full - length transgenic models of disease .
first , the exclusion of any intronic sequences in the atxn3 transcript of q135 may significantly alter rna processing dynamics of the mutant transcript , which could have direct effects on aso targeting capacity .
in addition , q135 mice express a single isoform of human atxn3 , the mrna structure of which may be less accessible to aso binding than other isoforms . in support of this argument ,
two previous studies investigating aso therapy for the polyglutamine disease spinal bulbar muscular atrophy ( sbma ) noted much less suppression of the targeted transcript in a cdna transgenic mouse model than in a mouse model of sbma expressing the full - length gene.33 , 34 future studies could include other biologically relevant models of sca3 such as recently available knock - in models to further validate aso potency .
we further assessed the tolerability of the three most effective asos in  vivo . aso-2 and
aso-5 proved to be safe and well tolerated , as evidenced by the absence of astrocytic or microglial activation after treatment .
aso-5 even decreased transcript levels of the microglia marker iba1 in q84 mice , suggesting a partial rescue of neuroinflammatory changes that occur in sca3 patient brains as well as in transgenic sca3 mouse models.22 , 36 in contrast to aso-2 and aso-5 , aso-4 caused significant elevation of gfap transcript levels in the diencephalon and increased astrocytic invasion into the dcn . although aso-4 was designed to have low off - targeting potential , the apparent toxicity in fibroblast and animal models of disease do not support further development of aso-4 as a potential therapeutic in sca3 .
importantly , the lack of an overt immune response following endogenous atxn3 suppression , either in the presence or absence of the human disease protein , supports previous findings that atxn3 is likely not an essential protein and that non - allele - specific silencing of atxn3 may be well tolerated as a therapeutic approach.7 , 8 future studies may employ additional in  vivo safety and toxicity evaluations , such as transcriptome analysis or stereology in treated sca3 mice .
the immediate next step is to move the safe and efficacious asos , aso-2 and aso-5 , into a long - term trial to assess their impact on behavioral phenotypes and animal survival .
while hemizygous q84 mice used in these studies express the full - length human atxn3 gene , they display relatively subtle behavioral phenotypes . for a long - term trial
, we will need to employ homozygous q84 mice , which recapitulate numerous aspects of the human disease including quantifiable motor deficits , robust aggregate pathology , and early lethality.3 , 23 in summary , we have established the first proof of concept for aso efficacy and tolerability in a sca3 mouse model of disease .
all animal procedures were approved by the university of michigan  committee on the use and care of animals ( ucuca ) .
yacmjd84.2q - c57bl/6 and cmvmjdq135-c57bl/6 transgenic mice were housed in cages with a maximum number of five animals and maintained in a standard 12-hr light / dark cycle with food and water ad libitum .
genotyping was performed using dna isolated from tail biopsy at the time of weaning , as previously described .
yacmjd84.2q transgenic mice hemizygosity was determined by standard pcr using gotaq green master mix ( promega ) to amplify a fragment of the atxn3 transgene with primer sequences 5-tggccttcacatggatgtgaa and 5-ccagtgactactttgattcg , and normalizing to a genomic fragment of mouse atxn3 chromosome 7 with primer sequences 5-ctctgtacagacagggagatgtgag and 5-gaggatgcaaaggagccaagtgacc .
bolus , anesthetized with 100  mg / kg ketamine per 10  mg / kg xylazine intraperitoneally , and perfused transcardially with pbs .
the five candidate anti - atxn3 asos and scrambled control aso  used in this study are 18 nucleotides in length with eight unmodified deoxynucleotides with a native sugar - phosphate backbone flanked by five moe - modified ribonucleotides on the 5- and 3-termini with a phosphorothioate backbone .
these asos were designed to target the intron , exon , or 3-utr of human atxn3 .
oligonucleotides were synthesized as described previously.37 , 38 asos were solubilized in pbs ( without ca or mg ) .
sca3 patient - derived fibroblasts ( gm06153 ) containing an expanded atxn3 allele ( 23q/71q ) were obtained from coriell cell repositories .
fibroblasts were maintained at 37c and 5% co2 in dmem ( hyclone ) supplemented with 15% fetal bovine serum .
hepg2 cells were obtained from atcc and maintained in eagle s minimum essential medium ( emem ) ( atcc ) supplemented with 10% fbs , 1% non - essential amino acid ( neaa ) , and 1% sodium pyruvate .
hepg2 cells were electroporated at 165  v in 100  l media with 20,000 cells / well on the btx high - throughput electroporation system ( harvard apparatus ) .
following electroporation , cells were transferred to a collagen - coated plate and placed in an incubator overnight .
twenty - four hours post - treatment , cells were washed with pbs and then lysed with gtc for rna purification .
sca3 patient - derived fibroblasts were plated into 12-well plates  at  80,000 cells / well density in supplemented dmem 2  days prior  to transfection .
aso transfections were performed using lipofectamine ltx with plus reagent ( life technologies ) at a final aso concentration of 4  m / well according to the manufacturer s instructions .
cells were harvested 48  hr after transfection in trizol ( invitrogen ) for rna analysis or 72  hr after  transfection using radioimmunoprecipitation assay ( ripa ) buffer containing protease inhibitors ( complete mini ; roche diagnostics ) for protein analysis .
total rna was isolated from transfected fibroblasts and dissected brain tissue using trizol reagent according to the manufacturer s protocol ( invitrogen ) .
reverse transcription was performed on 1  g total rna using the iscript cdna synthesis kit according to the manufacturer s instructions ( bio - rad ) .
iq sybr green qpcr was performed on the diluted cdna following the manufacturer s protocol ( bio - rad ) .
analysis was performed using average adjusted relative quantification using the following primers : human atxn3 ( 5-gaagctgaccaactcctgc-3 and 5-cttctaacactcgttccagg-3 ) , mouse beta actin ( 5-gaacggacagccatgggcggg-3 and 5-gtgtgtccccaagccccacg-3 ) , mouse gfap ( 5-gaaaaccgcatcaccattcc-3 and 5-cttattgacctcaccatcccg-3 ) , mouse iba1 ( 5- ccaccgtgtcagaatccac-3 and 5-atgctgggcaagagatct-3 ) , human gapdh ( 5-ctccgggtgatgcttttcct and 5-acatgtaaaccatgtagttgaggt ) , and mouse atxn3 ( 5-tgtcttgttacagaaagatcag and 5-gttacaagaacagagctgact ) .
protein lysates from sca3 fibroblasts were produced by lysis in ripa buffer containing protease inhibitors ( complete mini ) , followed by centrifugation .
protein lysates from the perfused and dissected mouse forebrain , diencephalon , cerebellum , and cervical spinal cord were produced by lysis and homogenization in ripa buffer containing protease inhibitors ( complete mini ) via dounce homogenizers , followed by sonication and centrifugation .
total protein concentrations of extracted supernatants were determined using the bicinchoninic acid ( bca ) method ( pierce ) and stored at 80c .
a total of 50  g total mouse brain protein lysate or 5  g total fibroblast protein lysate  was resolved in 10% sds - polyacrylamide electrophoresis gels and  transferred to polyvinylidene difluoride ( pvdf ) membranes .
membranes were incubated overnight at 4c with various antibodies : mouse anti - atxn3 ( 1h9 ) ( 1:1,000 , mab5360 ; millipore ) , rabbit anti - mjd antibody ( 1:10,000 ; paulson et  al . ) , mouse anti - gapdh ( 1:5,000 , mab374 ; millipore ) , or rabbit anti--tubulin ( 1:5,000 , s2144 ; cell signaling technology ) .
bound primary antibodies were  visualized by incubation with a peroxidase - conjugated anti - mouse or anti - rabbit secondary antibody ( 1:10,000 ; jackson immunoresearch laboratories ) followed by treatment with an enhanced chemiluminescence ( ecl)-plus reagent ( western lighting ; perkinelmer ) and exposure to autoradiography films .
six sex - matched q84 , q135 , or wild - type littermates were included per experimental treatment group .
for each injection , a small incision was made , the skull was exposed , and a small burr hole was drilled at the proper coordinates : anterior - posterior ,  + 0.3  mm ; mediolateral , 1.0  mm ; and dorsoventral , 3.0  mm relative to the bregma .
three minutes after the needle ( 7758 - 04 ; hamilton ) connected to a 10-l syringe ( 7653 - 01 ; hamilton ) was placed into the proper coordinates , a total of 500700  g aso diluted in 10  l pbs ( without ca or mg ) was delivered at an infusion rate of 0.5  l / s using an injection pump ( umc4 ; world precision instruments ) .
for all aso studies in q84 mice , mice received 500  g of the five active asos or control aso . in the q135 aso studies , mice received 500  g aso-4 , 700  g aso-5 , or 700  g aso - ctrl .
five minutes after the infusion was completed , the needle was retracted at a rate of 1  mm / s .
the incision site was sutured with synthetic absorbable sutures ( v1d397 ; vet one ) and the mouse was allowed to recover in a temperature - controlled environment . following surgery , weight , grooming activity , and home cage activity
whole brains perfused with pbs and the right hemispheres were post - fixed for 48  hr at 4c in 4% paraformaldehyde solution and immersed in 30% sucrose until saturated , and 40  m was sagittally sectioned on a sledge microtome ( sm200r ; leica biosystems ) .
brain sections were subjected to a basic antigen retrieval , washed , blocked , and incubated overnight at 4c in primary antibody supplemented with 0.025% triton x-100 , 0.5% bsa , and  5%  serum from the  host line for secondary antibodies ( donkey or goat ) .
primary antibodies used in these studies included the following : mouse anti - atxn3 ( 1h9 ) ( 1:1,000 , mab5360 ; millipore ) , rabbit anti - aso ( 1:5,000 , ionis pharmaceuticals ) , rabbit anti - neun-488 conjugated ( 1:1,000 , abn78a4 ; millipore ) , mouse anti - gfap ( 1:1,000 , 3670 ; cell signaling technology ) , and rabbit anti - iba1 ( 1:1,000 , 019 - 19741 ; wako chemicals ) .
primary incubated sections were then washed and incubated with the corresponding secondary alexa fluor 488 or 568 antibodies ( 1:1,000 ; invitrogen ) .
all sections were stained with dapi ( sigma ) for 15  min at room temperature , mounted with prolong gold antifade reagent ( invitrogen ) , and imaged using an ix71 olympus inverted microscope or nikon - a1 confocal microscope .
conceptualization , l.r.m , g.r . , g.h . , h.b.k . , h.l.p . , h.s.m .
 the most common dominantly inherited ataxia , spinocerebellar ataxia type 3 ( sca3 ) , is an incurable neurodegenerative disorder caused by a cag repeat expansion in the atxn3 gene that encodes an abnormally long polyglutamine tract in the disease protein , atxn3 . 
 mice lacking atxn3 are phenotypically normal ; hence , disease gene suppression offers a compelling approach to slow the neurodegenerative cascade in sca3 . 
 here we tested antisense oligonucleotides ( asos ) that target human atxn3 in two complementary mouse models of sca3 : yeast artificial chromosome ( yac ) mjd - q84.2 ( q84 ) mice expressing the full - length human atxn3 gene and cytomegalovirus ( cmv ) mjd - q135 ( q135 ) mice expressing a human atxn3 cdna . 
 intracerebroventricular injection of asos resulted in widespread delivery to the most vulnerable brain regions in sca3 . in 
 treated q84 mice , three of five tested  asos reduced disease protein levels by > 50% in the diencephalon , cerebellum , and cervical spinal cord . 
 two asos also significantly reduced mutant atxn3 in the mouse forebrain and resulted in no signs of astrogliosis or microgliosis . in q135 mice expressing a single atxn3 isoform via a cdna transgene 
 , asos did not result in similar robust atxn3 silencing . 
 our results indicate that asos targeting full - length human atxn3 would likely be well tolerated and could lead to a preventative therapy for sca3 . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest