antisense - mediated exon skipping has a lot of therapeutic potential for duchenne muscular dystrophy ( dmd ) , a severe , progressive neuromuscular disorder that affects one in 3,500 newborn boys .
this disease is caused by mutations , mostly deletions of one or more exons , in the dystrophin - encoding dmd gene that disrupt the reading frame and give rise to prematurely truncated proteins that are non functional .
dystrophin is crucial for maintaining muscle fiber stability and lack of dystrophin leads to exercise - induced damage and continuous loss of muscle fibers and function and eventually premature death usually in the 3rd decade .
the exon skipping approach aims to reframe the disrupted transcripts , allowing the production of internally deleted , but partially functional dystrophin proteins , as are found in becker muscular dystrophy patients .
this can be achieved with antisense oligonucleotides ( aons ) , small pieces of modified rna that specifically hybridize to a target exon , hide it from the splicing machinery causing it not to be included in the mrna .
this approach is mutation - specific , but since deletions cluster in a hotspot region , skipping some exons applies to larger groups of patients .
exon 51 skipping would be beneficial for the largest group of patients ( 13% ) and is currently developed in clinical trials .
initial trials using intramuscular injections of aons of two chemistries [ pro051/gsk 2402968 , a 2-o - methyl phosphorothioate ( 2omeps ) and avi-4658 , a phosphorodiamidate morpholino oligomer ( pmo ) ] resulted in local dystrophin restoration in the majority of muscle fibers at ~1535% of wild - type levels . both compounds have also been tested in systemic trials . for pro051/gsk2402968 ( 2omeps chemistry ) 5 weekly subcutaneous injections at different doses was well tolerated and induced novel dystrophin expression was observed in 60100% of muscle fibers in post - treatment biopsies of 10/12 dmd patients .
dystrophin levels increased in a dose - dependent manner to up to 15.6% of that in healthy muscle .
all 12 patients were subsequently enrolled in an open label extension study receiving weekly doses of 6  mg / kg .
after 12 weeks , a mean improvement of 35.2 m ( 28.7 ) was observed in the 6-minute walk test ( > 65 meters in three patients ) .
although these results are very encouraging , they will need to be confirmed by blinded and placebo - controlled follow up studies , which are currently ongoing .
weekly intravenous injections of avi-4658 ( pmo chemistry ) for 12 weeks were tested at doses up to 20  mg / kg .
this revealed dystrophin restoration in seven patients , especially for the higher dose groups . in three very good responders up to 55% of fibers became dystrophin positive and up to 27% of wild - type levels were observed . due to aon , dystrophin mrna transcript and protein turnover , lifelong repeated aon treatment will be required .
animal models have been instrumental in the optimization of systemic delivery of aons and assessing their pharmacodynamic and pharmacokinetic properties .
. however , the phenotype in mdx mice is less severe than in humans : mice have a more efficient regeneration , muscle function is only mildly impaired and lifespan nearly matches that of wild type mice . in mdx mice ,
due to the minimal phenotype , it is more difficult to establish functional efficacy in this model . in other animal models , such as dogs ,
. the mild phenotype in mdx mice might in part be due to overexpression of the dystrophin homologue utrophin .
indeed , mice that lack utrophin in addition to dystrophin ( double knockout mice , mdx / utrn ) have a very severe phenotype , with a limited lifespan ( generally 23 months ) .
it has been possible to prolong the lifespan of these animals to > 1 year using peptide - conjugated pmos .
however , due to the severity of this model , animals could only be rescued when treatment started before weaning at very high doses ; while treatment that was initiated later , or treatment with lower doses had no effect .
it has been reported that dystrophin - negative mice with a single utrophin allele ( mdx / utrn ) have an intermediate phenotype , with higher levels of fibrosis than mdx and more impaired respiratory function .
our own studies show that muscle function is also more impaired than in dystrophin - negative mice with two utrophin alleles .
thus , this model might be more suitable than traditional mdx mice for therapeutic studies , as it would allow a better assessment of the effect of exon skipping on muscle function and quality . toward long - term clinical trials we here tested weekly treatment with 2omeps aons at a high dose for a period of 6 months in mdx and mdx / utrn mice . in both models treatment
did induce exon skipping , restored dystrophin and improved muscle function and integrity , in the absence of toxic effects .
exon 23 skipping would bypass the nonsense mutation present in the dmd gene of the mdx and mdx / utrn mouse models .
we here used 2omeps aons targeting the 5 splice , previously identified by the group of steve wilton . for 2omeps aons , exon skipping after systemic aon treatment of mdx mice
can be achieved with a dose of 50  mg / kg / week , but the optimal dose appears to be 200  mg / kg / week , which would correspond to a dose of 16  mg / kg in humans after correcting for differences in body surface area between small and large animals ( http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm078932.pdf ) .
mdx and the more severe mdx / utrn mice were treated with weekly subcutaneous injections of 200  mg / kg aons or saline for 24 weeks starting at 4 weeks of age ( n = 7 per group ) .
treatment was well tolerated by all mice and body weights were similar for saline and aon - treated animals over time ( data not shown ) .
aon uptake by skeletal muscles , heart , diaphragm , liver , spleen , and kidney was assessed by an aon - specific ligation - hybridization assay ( figure 1a ) .
this revealed higher uptake by kidney for the mdx / utrn mice ( p value < 0.05 ) , whereas aon tissue levels were comparable for diaphragm , gastrocnemius , tibialis anterior , heart , and liver .
reverse transcription - pcr analysis of skeletal muscles , heart and diaphragm showed exon 23 skipping for all treated mice ( figure 1b ) , whereas no skipping was observed in saline - treated mice ( data not shown ) .
no significant differences were observed between skipping levels observed for mdx and mdx / utrn mice for any of the muscles analyzed ( figure 1c ) .
exon skipping levels in heart were much lower ( ~5% ) than in the other muscles ( ~15% ) , which has been reported before for 2omeps aons and pmos .
exon skipping also resulted in dystrophin restoration as assessed by western blot analysis ( figure 1d , e ) .
dystrophin levels in gastrosnemius muscles were comparable to those obtained after 3-month treatment with 2 weekly injections of 100  mg / kg in both mdx and mdx / utrn mice ( 23% of wild - type levels ) .
plasma creatine kinase ( ck ) levels are a marker for muscle fiber integrity and were assessed on a weekly basis .
ck levels in untreated mdx / utrn mice were higher over time than those of mdx mice ( figure 2a ) .
although the dystrophin levels observed in treated mice were modest , ck levels dropped very significantly in treated mice over time ( p value < 10 and 10 , for mdx and mdx / utrn , respectively ; figure 2b , c ) .
no significant difference was observed at any time point or over time in grip strength between the different models or between the treated and untreated groups ( data not shown ) . without treatment , the mdx mice clearly outperformed mdx / utrn mice in the rotarod analysis over time ( p value < 0.01 , figure 3a ) , corresponding to our previous results in younger mice . no difference could be observed between treated and untreated mdx mice ( figure 3b ) , whereas mdx / utrn - treated mice performed significantly better than untreated mice ( p value < 0.01 ; figure 3c ) . as described above , long - term subcutaneous aon treatment did not result in any obvious changes in body weight during treatment .
plasma was collected at the end of treatment and organs harvested for toxicological analysis , which did not reveal any toxic effects .
liver weights did not differ between treated and untreated mice ( figure 4a ) and bilirubin levels ( a marker for liver damage / inflammation ) were below the detection limit for all mice ( data not shown ) .
alp levels were increased in treated mice compared to untreated mice , although levels stayed within the normal range for all groups , but this difference was not significant ( p value cut - off 0.01 after multiple testing correction ) ( figure 4b ) .
glutamic oxaloacetic pyruvate transanimase and glutamate pyruvic transanimase are enzymes that are present in liver and muscle and leak into the bloodstream upon liver and muscle damage .
consequently , glutamic oxaloacetic pyruvate transanimase and glutamate pyruvic transanimase levels are elevated in dystrophic mouse models .
notably , levels in mdx mice are much higher than in the more severely affected mdx / utrn mouse
. this might be due to strain differences ( c57/bl10 versus c57/bl6 backgrounds for mdx and mdx / utrn , respectively ) .
levels did not further increase for treated mdx mice , while a nonsignificant increase was observed for treated mdx / utrn mice ( p value > 0.01 ) ( figure 4c , d ) . the kidney weight was similar for all groups ( figure 4e ) , as were urea levels ( a marker for kidney function ) ( figure 4f ) .
an increase in spleen weight could be observed for treated mdx mice , but this was not significant ( figure 4 g ) .
no difference in spleen weight was observed for mdx / utrn mice or in hemoglobin levels for both models ( figure 4h ) .
these results confirm that 6-month treatment with a high dose of 2omeps aon is well tolerated .
exon 23 skipping would bypass the nonsense mutation present in the dmd gene of the mdx and mdx / utrn mouse models .
we here used 2omeps aons targeting the 5 splice , previously identified by the group of steve wilton . for 2omeps aons , exon skipping after systemic aon treatment of mdx mice
can be achieved with a dose of 50  mg / kg / week , but the optimal dose appears to be 200  mg / kg / week , which would correspond to a dose of 16  mg / kg in humans after correcting for differences in body surface area between small and large animals ( http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm078932.pdf ) .
mdx and the more severe mdx / utrn mice were treated with weekly subcutaneous injections of 200  mg / kg aons or saline for 24 weeks starting at 4 weeks of age ( n = 7 per group ) .
treatment was well tolerated by all mice and body weights were similar for saline and aon - treated animals over time ( data not shown ) .
aon uptake by skeletal muscles , heart , diaphragm , liver , spleen , and kidney was assessed by an aon - specific ligation - hybridization assay ( figure 1a ) .
this revealed higher uptake by kidney for the mdx / utrn mice ( p value < 0.05 ) , whereas aon tissue levels were comparable for diaphragm , gastrocnemius , tibialis anterior , heart , and liver .
reverse transcription - pcr analysis of skeletal muscles , heart and diaphragm showed exon 23 skipping for all treated mice ( figure 1b ) , whereas no skipping was observed in saline - treated mice ( data not shown ) .
no significant differences were observed between skipping levels observed for mdx and mdx / utrn mice for any of the muscles analyzed ( figure 1c ) .
exon skipping levels in heart were much lower ( ~5% ) than in the other muscles ( ~15% ) , which has been reported before for 2omeps aons and pmos .
exon skipping also resulted in dystrophin restoration as assessed by western blot analysis ( figure 1d , e ) .
dystrophin levels in gastrosnemius muscles were comparable to those obtained after 3-month treatment with 2 weekly injections of 100  mg / kg in both mdx and mdx / utrn mice ( 23% of wild - type levels ) .
plasma creatine kinase ( ck ) levels are a marker for muscle fiber integrity and were assessed on a weekly basis .
ck levels in untreated mdx / utrn mice were higher over time than those of mdx mice ( figure 2a ) .
although the dystrophin levels observed in treated mice were modest , ck levels dropped very significantly in treated mice over time ( p value < 10 and 10 , for mdx and mdx / utrn , respectively ; figure 2b , c ) .
no significant difference was observed at any time point or over time in grip strength between the different models or between the treated and untreated groups ( data not shown ) . without treatment , the mdx mice clearly outperformed mdx / utrn mice in the rotarod analysis over time ( p value < 0.01 , figure 3a ) , corresponding to our previous results in younger mice . no difference could be observed between treated and untreated mdx mice ( figure 3b ) , whereas mdx / utrn - treated mice performed significantly better than untreated mice ( p value < 0.01 ; figure 3c ) .
as described above , long - term subcutaneous aon treatment did not result in any obvious changes in body weight during treatment .
plasma was collected at the end of treatment and organs harvested for toxicological analysis , which did not reveal any toxic effects .
liver weights did not differ between treated and untreated mice ( figure 4a ) and bilirubin levels ( a marker for liver damage / inflammation ) were below the detection limit for all mice ( data not shown ) .
alp levels were increased in treated mice compared to untreated mice , although levels stayed within the normal range for all groups , but this difference was not significant ( p value cut - off 0.01 after multiple testing correction ) ( figure 4b ) .
glutamic oxaloacetic pyruvate transanimase and glutamate pyruvic transanimase are enzymes that are present in liver and muscle and leak into the bloodstream upon liver and muscle damage . consequently , glutamic oxaloacetic pyruvate transanimase and glutamate pyruvic transanimase levels are elevated in dystrophic mouse models .
notably , levels in mdx mice are much higher than in the more severely affected mdx / utrn mouse
. this might be due to strain differences ( c57/bl10 versus c57/bl6 backgrounds for mdx and mdx / utrn , respectively ) .
levels did not further increase for treated mdx mice , while a nonsignificant increase was observed for treated mdx / utrn mice ( p value > 0.01 ) ( figure 4c , d ) .
the kidney weight was similar for all groups ( figure 4e ) , as were urea levels ( a marker for kidney function ) ( figure 4f ) .
an increase in spleen weight could be observed for treated mdx mice , but this was not significant ( figure 4 g ) .
no difference in spleen weight was observed for mdx / utrn mice or in hemoglobin levels for both models ( figure 4h ) .
these results confirm that 6-month treatment with a high dose of 2omeps aon is well tolerated .
antisense - mediated exon skipping is currently considered one of the most promising therapeutic approaches to treat dmd . due to aon , mrna transcript and dystrophin protein turnover , repeated treatment will be required .
aons are very small and are therefore generally cleared by the kidney . due to the ps backbone , 2omeps aons will bind to serum proteins , which prevents renal clearance and increases the half - life in serum and tissue . we have previously shown that 2omeps aons are taken up efficiently by dystrophic muscle and that body wide exon skipping and dystrophin restoration can be achieved after subcutaneous treatment for up to 3 months in mdx mice in the absence of toxic effects .
we here tested 6-month treatment with high doses ( 200  mg / kg / week ) in two different dystrophic mouse models , i.e. , the mdx and the slightly more severely affected mdx / utrn model . compared to the 6  mg / kg / week dose used in clinical trials for gsk2404968 , a 2omeps aon targeting exon 51 ,
however , a correction factor applies when translating doses between small and larger animals based on normalization to body surface area ( http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm078932.pdf ) . when applying this correction factor , a dose of 16  mg / kg would be predicted for humans , which is in the same order of magnitude as the 6  mg / kg used in the trials .
the slightly higher corrected dose for mice might be explained by a higher clearance rate for 2omeps aons in mice , potential differences between pharmacokinetic and pharmacodynamic properties between exon 23 and exon 51 targeting aons and the fact that a more efficient utrophin upregulation in mice leads to more stable muscle fibers and thus lower aon uptake .
we would like to propose the mdx / utrn model as an easier model than the mdx / utrn mouse , where breeding and husbandry is challenging .
still , the disease is more severe in the mdx / utrn model than in the traditionally used mdx mouse model .
higher levels of fibrosis and a more impaired respiratory function have been reported by others and we recently reported that the mdx / utrn model also performs worse than mdx mice in muscle function tests .
thus , assessment of treatment effects on muscle function can be easier picked up in this model .
indeed , while in untreated animals ck levels and rotarod running times were significantly more affected in mdx / utrn mice , they were comparable for mdx and mdx / utrn after 6 months aon treatment .
thus , the same amount of dystrophin was able to achieve a larger therapeutic effect in the more severe mdx / utrn mice .
while there is a clear benefit of these low dystrophin levels on muscle function and quality , it should be stressed that they do not fully normalize the phenotype .
we have recently shown that levels of ~1520% are probably needed for a more complete normalization of the phenotype in a mouse model with variable , low dystrophin levels due to skewed x - inactivation .
interestingly , we were unable to find a difference between treated and untreated mdx mice in rotarod running after 24-week treatment , while we have previously shown improvement in rotarod running in mdx mice after 8 weeks of aon treatment . as the analysis is done over time , a possible explanation for this discrepancy is the different duration of the experiments .
aon levels in muscles were comparable between the mouse models and highest in heart , while skipping levels were lowest for this tissue .
however , the hybridization - ligation assay detects aon levels in the complete tissue and can not discriminate between aon in the tissue and aon trapped in the interstitium .
based on the lower exon skipping levels observed in heart , we hypothesize more aons are trapped in the interstitium for heart than skeletal muscles .
lower exon skipping levels in heart have been reported previously after systemic 2omeps and pmo treatment , probably because the heart cells are not leaky like the skeletal muscle fibers and thus do not benefit from an enhanced uptake of aons . when exon skipping and dystrophin levels in mdx mice were compared to those obtained after 4 , 8 , and 12 weeks of treatment ,
no further accumulation was observed , while an increase had been observed previously up to 12 weeks .
this might be explained by a different dosing regime that was adopted to reduce stress and handing for the mice .
rather than 2 weekly subcutaneous injections of 100  mg / kg , these mice received 1 weekly injection of 200  mg / kg . it has recently been shown that for pmos multiple low doses are more efficacious than a single high dose .
even though dystrophin levels were quite low in treated mice , significant improvements could be observed in ck levels for both strains and in rotarod running time for the mdx / utrn mice .
it has been reported previously that very low amounts of dystrophin have an effect on muscle quality .
this might be related to additional functions of the dystrophin protein in signal transduction and regulating mirna levels .
there were some slight increases in some markers for liver function and liver damage , but none were significant .
in addition , the differences were generally only found for one strain ( either mdx or mdx / utrn ) . this suggests that 6-month treatment with high doses of 2omeps aon by subcutaneous injections is well tolerated .
aons have thus far primarily been used to treat acute diseases and consequently , long - term safety data in humans is not yet available .
therefore , patients will have to be carefully monitored during long - term clinical trials that are currently ongoing for exon 51 skipping . in conclusion , we show here that long - term subcutaneous treatment with 2omeps aons leads to exon skipping , dystrophin restoration , improved muscle quality and function in two dmd mouse models , in the absence of toxicity .
mdx / utrn and mdx mice ( n = 7 per group ) were treated with weekly subcutaneous injections of 200  mg / kg m23d ( + 218 ) a 2-o - methyl phosphorothioate aon described previously or saline .
ten days after the last injection mice were sacrificed by cervical dislocation and muscles ( gastrocnemius , quadriceps , tibialis anterior , triceps , heart , and diaphragm ) and organs ( liver , kidney , and spleen ) were isolated and snap frozen in liquid nitrogen cooled 2-methylbutane and stored at 80  c .
the assay for measuring the aon concentration in tissue samples is based on a previously published hybridization - ligation assay .
all tissues and calibration curves of the analyzed aon were diluted 60 times in pooled control mdx mouse tissue lysates of the same type .
the muscle samples were diluted 500 and 1,000 times , liver and kidney tissue 1,000 and 5,000 times .
muscles were minced in rna - bee ( campro scientific , veenendaal , the netherlands ) using magna lyser green beads ( roche diagnostics , almere , the netherlands ) and the magna lyser ( roche diagnostics ) according to the manufacturer 's instructions .
total rna was extracted and reverse transcription - pcr analysis was performed as described with a single round of pcr .
pcr products were visualized on 2% agarose gels and quantified with the agilent 2100 bioanalyzer ( agilent , amstelveen , the netherlands ) . protein extraction and western blot .
hcl ph 6.8 and 20% sodium dodecyl sulfate ) using magna lyser green beads in the magna lyser ( roche diagnostics ) .
protein concentration was determined using the bca protein assay kit ( thermo scientific , etten leur , the netherlands ) , according to the manufacturer 's instructions .
subsequently , the homogenate was completed to contain 75  mol / l tris  hcl ph 6.8 , 15% sodium dodecyl sulfate , 5% -mercaptoethanol , 20% glycerol , and 0.001% bromophenol blue and boiled for 5 minutes .
one hundred microgram was loaded on a 47% gradient polyacrylamide gel and run overnight at 4  c .
gels were blotted to nitrocellulose ba83 ( whatman , schleicher & schuell , germany ) for 6 hours at 4  c .
blots were blocked with 5% non - fat dried milk ( campina melkunie , maasdam , the netherlands ) in tbs followed by an overnight incubation with ncl - dys1 ( dilution 1:125 ; novacastra , leica microsystems , valkerswaard , the netherlands ) in tbs plus 0.05% tween-20 to detect dystrophin .
the fluorescent irdye 800cw goat - anti - mouse igg ( dilution 1:5,000 ; li - cor , lincoln , ne ) was used as a secondary antibody .
blots were visualized and quantified with the odyssey system and software ( li - cor ) .
-actinin ( ab72592 ; abcam , cambridge , ma , dilution 1:7,500 ) , was used as a loading control .
blood was collected weekly on monday mornings in a minicollect tube ( 0.8-ml lithium heparin sep ; greiner bio - one , alphen aan den rijn , the netherlands ) via a small cut at the end of the tail .
ck levels were determined in plasma with reflotron ck test strips in the reflotron plus machine ( roche diagnostics ) . immediately before scarifying , blood was collected to assess plasma hemoglobin , alp , urea , glutamic oxaloacetic transaminase , and glutamate pyruvate transaminase levels using the respective reflotron test strips . functional analyses .
forelimb grip strength was assessed by means of a grip strength meter ( columbus instruments , columbus , oh ) .
mice were tested five times , with three consecutive measurements per trial ( 15 in total ) , and a 2-minute interval between trials .
the three highest measured values were averaged to calculate the absolute strength , which was divided by the body weight in grams .
a rotarod test was performed weekly with the rotarod ( ugo basile , comerio , italy ) .
mice were placed on the rod that accelerated from 5 to 45 rotations per minute within 15 seconds .
when a mouse ran for 500 seconds without falling from the rod , the test session was ended .
mice that fell off within 500 seconds were given a maximum of two more tries .
a two - tailed homoscedastic student 's t - test was performed in microsoft office excel 2003 to compare aon tissue concentrations , exon skip levels , organ weights and plasma parameters between groups of mice .
a p value < 0.05 was considered significant for all tests , however for the analyses shown in figure 4 a bonferroni correction was applied for multiple testing and a p value < 0.01 was considered significant . to assess whether ck levels , forelimb grip strength or rotarod running times differed significantly over time between groups linear regression analysis was performed with statistical software in r ( version r.2.11.1 ) , using either mouse strain or treatment as variables .
data from week 0 were excluded from the analysis assessing a potential effect of treatment as this time point was before treatment .
mdx / utrn and mdx mice ( n = 7 per group ) were treated with weekly subcutaneous injections of 200  mg / kg m23d ( + 218 ) a 2-o - methyl phosphorothioate aon described previously or saline .
ten days after the last injection mice were sacrificed by cervical dislocation and muscles ( gastrocnemius , quadriceps , tibialis anterior , triceps , heart , and diaphragm ) and organs ( liver , kidney , and spleen ) were isolated and snap frozen in liquid nitrogen cooled 2-methylbutane and stored at 80  c .
the assay for measuring the aon concentration in tissue samples is based on a previously published hybridization - ligation assay .
all tissues and calibration curves of the analyzed aon were diluted 60 times in pooled control mdx mouse tissue lysates of the same type .
the muscle samples were diluted 500 and 1,000 times , liver and kidney tissue 1,000 and 5,000 times .
muscles were minced in rna - bee ( campro scientific , veenendaal , the netherlands ) using magna lyser green beads ( roche diagnostics , almere , the netherlands ) and the magna lyser ( roche diagnostics ) according to the manufacturer 's instructions .
total rna was extracted and reverse transcription - pcr analysis was performed as described with a single round of pcr .
pcr products were visualized on 2% agarose gels and quantified with the agilent 2100 bioanalyzer ( agilent , amstelveen , the netherlands ) . protein extraction and western blot .
hcl ph 6.8 and 20% sodium dodecyl sulfate ) using magna lyser green beads in the magna lyser ( roche diagnostics ) .
protein concentration was determined using the bca protein assay kit ( thermo scientific , etten leur , the netherlands ) , according to the manufacturer 's instructions .
subsequently , the homogenate was completed to contain 75  mol / l tris  hcl ph 6.8 , 15% sodium dodecyl sulfate , 5% -mercaptoethanol , 20% glycerol , and 0.001% bromophenol blue and boiled for 5 minutes .
one hundred microgram was loaded on a 47% gradient polyacrylamide gel and run overnight at 4  c .
gels were blotted to nitrocellulose ba83 ( whatman , schleicher & schuell , germany ) for 6 hours at 4  c .
blots were blocked with 5% non - fat dried milk ( campina melkunie , maasdam , the netherlands ) in tbs followed by an overnight incubation with ncl - dys1 ( dilution 1:125 ; novacastra , leica microsystems , valkerswaard , the netherlands ) in tbs plus 0.05% tween-20 to detect dystrophin .
the fluorescent irdye 800cw goat - anti - mouse igg ( dilution 1:5,000 ; li - cor , lincoln , ne ) was used as a secondary antibody .
blots were visualized and quantified with the odyssey system and software ( li - cor ) .
-actinin ( ab72592 ; abcam , cambridge , ma , dilution 1:7,500 ) , was used as a loading control .
blood was collected weekly on monday mornings in a minicollect tube ( 0.8-ml lithium heparin sep ; greiner bio - one , alphen aan den rijn , the netherlands ) via a small cut at the end of the tail .
ck levels were determined in plasma with reflotron ck test strips in the reflotron plus machine ( roche diagnostics ) . immediately before scarifying , blood was collected to assess plasma hemoglobin , alp , urea , glutamic oxaloacetic transaminase , and glutamate pyruvate transaminase levels using the respective reflotron test strips . functional analyses .
forelimb grip strength was assessed by means of a grip strength meter ( columbus instruments , columbus , oh ) .
mice were tested five times , with three consecutive measurements per trial ( 15 in total ) , and a 2-minute interval between trials .
the three highest measured values were averaged to calculate the absolute strength , which was divided by the body weight in grams .
a rotarod test was performed weekly with the rotarod ( ugo basile , comerio , italy ) .
mice were placed on the rod that accelerated from 5 to 45 rotations per minute within 15 seconds .
when a mouse ran for 500 seconds without falling from the rod , the test session was ended .
mice that fell off within 500 seconds were given a maximum of two more tries .
a two - tailed homoscedastic student 's t - test was performed in microsoft office excel 2003 to compare aon tissue concentrations , exon skip levels , organ weights and plasma parameters between groups of mice .
a p value < 0.05 was considered significant for all tests , however for the analyses shown in figure 4 a bonferroni correction was applied for multiple testing and a p value < 0.01 was considered significant . to assess whether ck levels , forelimb grip strength or rotarod running times differed significantly over time between groups linear regression analysis was performed with statistical software in r ( version r.2.11.1 ) , using either mouse strain or treatment as variables .
data from week 0 were excluded from the analysis assessing a potential effect of treatment as this time point was before treatment .
 antisense - mediated exon skipping for duchenne muscular dystrophy ( dmd ) is currently tested in phase 3 clinical trials . 
 the aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts , allowing the synthesis of a partly functional dystrophin protein . 
 studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides ( aons ) . here , we tested the safety and efficacy of subcutaneously administered 2-o - methyl phosphorothioate aon at 200  mg / kg / week for up to 6 months in mouse models with varying levels of disease severity : mdx mice ( mild phenotype ) and mdx mice with one utrophin allele ( mdx / utrn+/ ; more severe phenotype ) . 
 long - term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals . 
 notably , in the more severely affected mdx / utrn+/ mice the therapeutic effect was larger : creatine kinase ( ck ) levels were more decreased and rotarod running time was more increased . 
 this suggests that the mdx / utrn+/ model may be a more suitable model to test potential therapies than the regular mdx mouse . 
 our results also indicate that long - term subcutaneous treatment in dystrophic mouse models with these aons is safe and beneficial . 
Introduction
Results
AON uptake and exon in mdx and mdx/utrn
Long-term AON treatment leads to improved muscle quality and function
Long-term treatment with 2OMePS AONs is well tolerated
Discussion
Materials and Methods
All animal experiments were approved by the Animal Ethics Committee of the Leiden University Medical Center.