a defining neuropathologic feature of alzheimer 's disease ( ad ) , progressive supranuclear palsy , frontotemporal lobar dementia - tau type ( ftld - t ) , and some other neurodegenerative disorders is abnormal intracellular aggregates of tau protein , as neurofibrillary tangles and in some cases , glial tangles . collectively , these disorders are known as tauopathies .
highly penetrant missense mutations in tau - encoding mapt gene cause ftld - t proving that aggregated tau is pathogenic . in vitro experiments show that these mutations increase aggregation rates .
others are intronic , altering the alternative splicing of exon 10 , and changing the isoform ratios of tau protein .
also , common genetic variations in the mapt genomic region increase risk for developing progressive supranuclear palsy and parkinson 's disease . thus , altered tau protein or its regulation can cause neurodegeneration .
mapt knock - out mice are viable and mostly normal showing limited changes in axonal structure , muscle strength , and behavior .
because these studies were performed in mice completely lacking tau , the observed changes could be due to developmental effects of no tau at conception . in mice
, tau can be replaced in part by another microtubule - associated protein , map1b . whether this is true in humans is not known .
mouse tauopathy models where the human tau cdna sequence with an ftld - t mutation is overexpressed develop aggregated tau pathology .
studies in these models show that injection of aggregated tau causes tau aggregation at , and beyond the injection site .
thus , in tauopathies , tau aggregation may spread from cell to cell with seeding of new aggregates dependent on cytoplasmic tau concentrations .
ad mouse models where the amyloid precursor protein is overexpressed develop amyloid plaques , learning deficits , hyperactivity , long - term potentiation deficits , and axonal transport defects , but no neurofibrillary tangles .
these deficits in amyloid precursor protein transgenic mice are ameliorated in animals lacking endogenous tau .
one strategy for altering gene expression and the synthesis of a specific protein is the use of modified oligonucleotides complementary to sequences in a target gene that interfere with rna transcription , processing , or translation .
antisense oligonucleotide approaches are being used in trials to treat duchenne muscular dystrophy ( dmd ) .
this approach involves designing oligonucleotides to induce skipping of exons that contain a premature stop codon .
these oligonucleotides target intron  exon boundary splice site sequences and presumably act by blocking access of splicing factors to the targeted splice site .
when a mutated exon is skipped , the resulting protein is sufficiently active for improved muscle function .
antisense oligonucleotides that induce skipping of exon 51 in dystrophin transcripts are in phase 2/3 clinical trials .
preliminary results indicate exon skipping is achieved , and that muscle function is improved , although one of these trials failed to show efficacy . here , we tested modified oligonucleotides to reduce mapt mrna expression and tau protein levels .
structural changes include morpholine rings linked through phosphodiamidate bonds to the nitrogen moieties of nucleic acids .
oligonucleotides complementary to the sense strand of mapt were tested in the human neuroblastoma cell lines imr32 and sh - sy5y to identify candidate targets for treatment of tauopathies . within mapt , we targeted sequences bracketing the start codon , splice acceptors and donors , splicing branch points , polypyrimidine track - related sequences , and splicing enhancer and inhibitor sequences .
oligonucleotides that induce exon 1 skipping will result in an mrna without the start atg , and thus mapt mrna would not be translated .
oligonucleotides that induce exon skipping of exons 1 , 5 , or 7 would result in a frame shift and a downstream premature stop codon , resulting in nonsense - mediated decay , a reduction in mapt mrna , and a reduction in tau protein .
we also show that the latter oligonucleotide is able to reduce human tau mrna and protein levels in muscle in a mouse transgenic for the entire human mapt gene .
this work shows the feasibility of using morpholino oligonucleotides to reduce human tau expression in vivo .
we designed morpholino oligonucleotides complementary to mapt transcripts with the goal of reducing the amount of tau protein produced by cells ( table 1 ; figure 1 ) .
mapt exons 0 , 1 , 4 , 5 , 7 , 9 , and 10 were targeted .
exons 0 and 1 contain the 5 untranslated sequences of mapt mrna . the translation initiation atg is in exon 1 .
exons 1 , 4 , 5 , 7 , and 9 are constitutive exons found in all six brain mapt isoforms .
we also targeted exon 10 , an alternatively spliced exon present in three of the six brain isoforms . when exon 10 is included in mapt transcripts ,
when exon 10 is missing , the protein produced has only 3 repeats ( 3r tau ) .
mutations that increase exon 10 inclusion cause frontotemporal dementia  tau type . since excess 4r tau may be toxic , we tested oligonucleotides that would potentially block exon 10 inclusion ( e10.1e10.8 ) .
most oligonucleotides tested were designed to induce exon skipping , either by hybridizing to intronic branch point sequences ( e1.7 , e4.2 , e4.3 , e5.4 ) , or by targeting splice sites directly , bracketing intronic and exonic sequences ( e.g. , e1.1 ) . e9.1 and e9.2 targeted exon splicing enhancers in exon 9 , a constitutively spliced exon .
alternative splicing of exon 10 is controlled by exonic splicing enhancer and silencer elements and by intronic sequences downstream of the 3 end of the exon .
exonic splicing regulatory sequences were targeted by e10.3 , which is complementary to an sc-35-like splicing enhancer element and e10.4 , which is complementary to both an a / c rich splicing enhancer element and an exonic polypurine enhancer ( table 1 ) .
splicing regulatory sequences downstream of e10 include an intronic splicing silencer and an intron splicing modulator .
these elements were targeted by e10.5 , e10.6 , e10.7 , and e10.8 ( ref .
oligonucleotide e1.2 was designed to block translation by hybridizing to the atg start codon and surrounding sequence in exon 1 .
we initially used a 96 well format to test pairs of oligonucleotides targeting more than one sequence for a given exon .
human neuroblastoma cell lines sh - sy5y and imr32 were nucleofected with the oligonucleotides and harvested after 6 days .
mapt mrna levels were measured by reverse transcription quantitative real - time polymerase chain reaction ( rt - pcr ) .
tau protein , the product of the mapt gene , was measured by an enzyme - linked immunosorbent assay ( elisa ) and by immunoblotting .
both cell lines produce predominantly 3r tau with 4r mrna accounting for 13.323.5% of the total mapt mrna produced ( supplementary table s1 ) as previously observed with these cell lines .
oligonucleotides targeting exons 0 , 4 , 9 , and 10 did not significantly reduce transcript levels ( supplementary figures s1s4 ) . as described below , some of the oligonucleotides targeting exons 1 , 5 , and 7 did significantly reduce mapt transcript and tau protein levels .
we examined the effects of seven antisense morpholino oligonucleotides that target exon1 in imr32 cells ( figure 2b ) .
oligonucleotides tested include e1.7 , which is complementary to the exon 1 branch point and polypyrimidine tract sequences , e1.1 , which spans the splice acceptor sequence at the 5-end of the exon and the atg initiation codon , and e1.3e1.6 , targeting the splice donor sequence at the 3-end of the exon .
we also tested an oligonucleotide which did not hybridize to a splice site but that was complementary to the atg start codon and would potentially block translation ( e1.2 ) . when tested individually , only oligonucleotides that targeted the splice donor site significantly reduced mapt transcript levels .
the most potent was e1.4 , which is complementary to the last 7 nucleotides of exon 1 and the first 18 nucleotides of intron 1 .
this oligonucleotide reduced mapt mrna expression in imr32 cultures by 50% ( sem = 4.13% , n = 4 ) .
we also tried a combination of oligonucleotides that targeted the splice donor ( e1.4 ) and the splice acceptor site along with the atg codon ( e1.1 ) , or the branchpoint sequence e1.7 , or all three together .
we sequenced rt - pcr products from both imr32 and sh - sy5y cells ( supplementary figures s5 and s6 ) .
the sequence from mock - treated cells ( no oligonucleotide control ) showed that exon 1 was spliced to exon 4 and alternatively spliced exons 2 and 3 were excluded ( 0n tau ) .
rt - pcr products from cells treated either with e1.4 alone or e1.1 + e1.4 showed two products containing exon 0 and exon 4 ( supplementary figure s6 ) .
the second smaller products showed exon 0 spliced directly to exon 4 , consistent with the skipping of exon 1 .
this latter product is consistent with a transcript lacking the atg codon and would not be expected to produce tau protein . when total tau protein was measured using an elisa , e1.4 reduced protein levels by 77% ( tau protein in  no - oligo 
pg tau/g protein in e1.4-treated cultures ; n = 5 ) in imr32 and 48% ( 88.24    8.5
pg tau/g protein versus 45.7    13.2 pg tau/g protein ; n = 3 ) in sh - sy5y cells ( figure 2d , e ) .
these morpholinos were complementary to the branchpoint sequence ( e5.4 ) , the splice acceptor site ( e5.1 ) , and the splice donor site ( e5.2 and e5.3 ) ( table 1 ) .
only e5.3 , when tested individually , significantly reduced total mapt transcript levels in both cell types tested to 28.546% ( figure 3a , b ) .
this morpholino was complementary to the last 6 nucleotides of exon 5 and the first 19 nucleotides of intron 5 .
transcript levels were also significantly reduced when both the acceptor and donor sequences were targeted ( e5.1 + e5.2 ) .
e5.3 also reduced total tau protein levels in imr32 cells ( 62% ; 357.6    37
pg tau/g protein versus 133    29 pg tau/g protein ; n = 3 ) and in sh - sy5y cells ( 58% , 88.24    8.5
mol / l , the highest concentration tested , though some reduction was observed with 5 and 1 mol / l ( supplementary figure s7 ) .
we sequenced rt - pcr products from both imr32 and sh - sy5y cells treated with a combination of e5.1 and e5.2 .
the second smaller sized product showed exon 4 spliced to exon 7 ( supplementary figure s8a , b ) indicating that e5.1 and e5.2 blocked inclusion of exon 5 into the final mapt transcript .
the same results were observed in cells treated with e5.3 oligo ( supplementary figure s8c ) .
we tested three morpholino oligonucleotides targeting the splice acceptor ( e7.1 , e7.2 ) and the splice donor site ( e7.3 ) ( supplementary figure s9 ) .
these oligonucleotides did not significantly reduce mapt transcript levels in imr32 cells ( supplementary figure s9a ) , but e7.3 did in sh - sy5y cells by 30% ( supplementary figure s9b ; sem = 5% , n = 3 ) .
e7.1 targets the exon 7 splice acceptor site and is complementary to the last 2 nucleotides of intron 6 and the first 23 nucleotides in exon 7 ( table 1 ) .
e7.3 did reduce protein expression in imr32 cells , but the difference was not significant ( supplementary figure s9c ; p = 0.07 ) .
however , in sh - sy5y cultures , consistent with decrease in mapt transcripts , e7.3 oligonucleotide also decreased tau protein levels by 67% ( supplementary figure s9d ; 88.24    8.5
pg tau/g versus 29    10.2 pg tau/g ; n = 3 ; p = 0.005 ) .
exon 10 is alternatively spliced and is found in three of the six tau isoforms found in brain .
none of the oligonucleotides directly targeting exon 10 reduced total mapt mrna levels nor did they alter the amount of exon 10 + ( 4r ) message ( supplementary figure s4 )
. we also examined exon 10 inclusion in sh - sy5y cells using morpholinos that targeted exons 1 , 4 , and 5 ( figure 4 ) . for exon 1 ,
oligonucleotides e1.4 and e1.1 + e1.4 that reduced total mapt transcript levels ( figure 2a , b ) reduced both 3r and 4r transcripts equally ( figure 4a , b ) .
the same was true for exon 5 oligonucleotides e5.3 and e5.1 , which reduced total mapt transcript levels ( figure 3a , b ) and transcripts encoding 3r and 4r isoforms ( figure 4c , d ) .
morpholinos masking polypyrimidine track ( e4.2 and e4.3 ) , as well as additional oligo targeting the splice donor site , e4.5 , also did not cause an appreciable reduction in total transcript levels ( data not shown ) .
oligonucleotides targeting the splice acceptor and splice donor sequences to exon 4 , e4.1 and e4.4 , respectively ( table 1 ) did not significantly reduce total mapt transcript levels ( supplementary figure s3 ) though there was a trend toward reduced expression .
however , two rt - pcr products were evident in cells treated simultaneously with e4.1 and e4.4 , and when we sequenced the smaller product , exon 1 was spliced to exon 5 ( supplementary figure s10 ) , the expected result if exon 4 were skipped and alternatively spliced exons 2 and 3 were also skipped .
interestingly , this pair of oligonucleotides reduced 4r but not 3r transcripts ( figure 4e , f ) .
presumably , we did not observe a reduction in total mapt transcript levels with e4.1 + e4.4 because the 4r isoform is only a minor component of the total mapt produced ( supplementary table s1 ) .
to determine whether anti - mapt oligonucleotides can affect gene expression in vivo , we used mice transgenic for the human mapt gene .
the transgene used is a 201  kb fragment of human genomic dna that contains the entire mapt gene and some flanking sequence .
the mice produce all six isoforms of the human tau protein and are null for the mouse mapt gene .
we injected the gastrocnemius muscles with either e5.3 or the control oligonucleotide or no injection .
histological analysis showed that these injections caused minimal tissue damage ( supplementary figure s11 ) .
we found that muscles ( left ) injected with e5.3 showed significantly less mapt mrna compared to noninjected muscles .
noninjected muscles had comparable levels of mrna and protein when compared to the control oligonucleotide ( figure 5a , b ) . e5.3
reduced tau protein from 18.1    3.6 to 5.3    0.85 pg tau/g protein ( p = 0.02 , figure 5c , d ) .
these results are comparable to those observed in neuroblastoma cell lines ( figure 3 ) .
oligonucleotides targeting exons 0 , 4 , 9 , and 10 did not significantly reduce transcript levels ( supplementary figures s1s4 ) . as described below , some of the oligonucleotides targeting exons 1 , 5 , and 7 did significantly reduce mapt transcript and tau protein levels .
we examined the effects of seven antisense morpholino oligonucleotides that target exon1 in imr32 cells ( figure 2b ) .
oligonucleotides tested include e1.7 , which is complementary to the exon 1 branch point and polypyrimidine tract sequences , e1.1 , which spans the splice acceptor sequence at the 5-end of the exon and the atg initiation codon , and e1.3e1.6 , targeting the splice donor sequence at the 3-end of the exon .
we also tested an oligonucleotide which did not hybridize to a splice site but that was complementary to the atg start codon and would potentially block translation ( e1.2 ) . when tested individually , only oligonucleotides that targeted the splice donor site significantly reduced mapt transcript levels .
the most potent was e1.4 , which is complementary to the last 7 nucleotides of exon 1 and the first 18 nucleotides of intron 1 .
this oligonucleotide reduced mapt mrna expression in imr32 cultures by 50% ( sem = 4.13% , n = 4 ) .
we also tried a combination of oligonucleotides that targeted the splice donor ( e1.4 ) and the splice acceptor site along with the atg codon ( e1.1 ) , or the branchpoint sequence e1.7 , or all three together .
we sequenced rt - pcr products from both imr32 and sh - sy5y cells ( supplementary figures s5 and s6 ) .
the sequence from mock - treated cells ( no oligonucleotide control ) showed that exon 1 was spliced to exon 4 and alternatively spliced exons 2 and 3 were excluded ( 0n tau ) .
rt - pcr products from cells treated either with e1.4 alone or e1.1 + e1.4 showed two products containing exon 0 and exon 4 ( supplementary figure s6 ) .
the second smaller products showed exon 0 spliced directly to exon 4 , consistent with the skipping of exon 1 .
this latter product is consistent with a transcript lacking the atg codon and would not be expected to produce tau protein . when total tau protein was measured using an elisa , e1.4 reduced protein levels by 77% ( tau protein in  no - oligo 
pg tau/g protein in e1.4-treated cultures ; n = 5 ) in imr32 and 48% ( 88.24    8.5
pg tau/g protein versus 45.7    13.2 pg tau/g protein ; n = 3 ) in sh - sy5y cells ( figure 2d , e ) .
these morpholinos were complementary to the branchpoint sequence ( e5.4 ) , the splice acceptor site ( e5.1 ) , and the splice donor site ( e5.2 and e5.3 ) ( table 1 ) .
only e5.3 , when tested individually , significantly reduced total mapt transcript levels in both cell types tested to 28.546% ( figure 3a , b ) .
this morpholino was complementary to the last 6 nucleotides of exon 5 and the first 19 nucleotides of intron 5 .
transcript levels were also significantly reduced when both the acceptor and donor sequences were targeted ( e5.1 + e5.2 ) .
e5.3 also reduced total tau protein levels in imr32 cells ( 62% ; 357.6    37
pg tau/g protein versus 133    29 pg tau/g protein ; n = 3 ) and in sh - sy5y cells ( 58% , 88.24    8.5
mol / l , the highest concentration tested , though some reduction was observed with 5 and 1 mol / l ( supplementary figure s7 ) .
we sequenced rt - pcr products from both imr32 and sh - sy5y cells treated with a combination of e5.1 and e5.2 .
the second smaller sized product showed exon 4 spliced to exon 7 ( supplementary figure s8a , b ) indicating that e5.1 and e5.2 blocked inclusion of exon 5 into the final mapt transcript .
the same results were observed in cells treated with e5.3 oligo ( supplementary figure s8c ) .
we tested three morpholino oligonucleotides targeting the splice acceptor ( e7.1 , e7.2 ) and the splice donor site ( e7.3 ) ( supplementary figure s9 ) .
these oligonucleotides did not significantly reduce mapt transcript levels in imr32 cells ( supplementary figure s9a ) , but e7.3 did in sh - sy5y cells by 30% ( supplementary figure s9b ; sem = 5% , n = 3 ) .
e7.1 targets the exon 7 splice acceptor site and is complementary to the last 2 nucleotides of intron 6 and the first 23 nucleotides in exon 7 ( table 1 ) .
e7.3 did reduce protein expression in imr32 cells , but the difference was not significant ( supplementary figure s9c ; p = 0.07 ) .
however , in sh - sy5y cultures , consistent with decrease in mapt transcripts , e7.3 oligonucleotide also decreased tau protein levels by 67% ( supplementary figure s9d ; 88.24    8.5 pg tau/g versus 29    10.2 pg tau/g ; n = 3 ; p = 0.005 ) .
exon 10 is alternatively spliced and is found in three of the six tau isoforms found in brain .
none of the oligonucleotides directly targeting exon 10 reduced total mapt mrna levels nor did they alter the amount of exon 10 + ( 4r ) message ( supplementary figure s4 ) .
we also examined exon 10 inclusion in sh - sy5y cells using morpholinos that targeted exons 1 , 4 , and 5 ( figure 4 ) . for exon 1 ,
oligonucleotides e1.4 and e1.1 + e1.4 that reduced total mapt transcript levels ( figure 2a , b ) reduced both 3r and 4r transcripts equally ( figure 4a , b ) .
the same was true for exon 5 oligonucleotides e5.3 and e5.1 , which reduced total mapt transcript levels ( figure 3a , b ) and transcripts encoding 3r and 4r isoforms ( figure 4c , d ) .
morpholinos masking polypyrimidine track ( e4.2 and e4.3 ) , as well as additional oligo targeting the splice donor site , e4.5 , also did not cause an appreciable reduction in total transcript levels ( data not shown ) .
oligonucleotides targeting the splice acceptor and splice donor sequences to exon 4 , e4.1 and e4.4 , respectively ( table 1 ) did not significantly reduce total mapt transcript levels ( supplementary figure s3 ) though there was a trend toward reduced expression .
however , two rt - pcr products were evident in cells treated simultaneously with e4.1 and e4.4 , and when we sequenced the smaller product , exon 1 was spliced to exon 5 ( supplementary figure s10 ) , the expected result if exon 4 were skipped and alternatively spliced exons 2 and 3 were also skipped .
interestingly , this pair of oligonucleotides reduced 4r but not 3r transcripts ( figure 4e , f ) .
presumably , we did not observe a reduction in total mapt transcript levels with e4.1 + e4.4 because the 4r isoform is only a minor component of the total mapt produced ( supplementary table s1 ) .
to determine whether anti - mapt oligonucleotides can affect gene expression in vivo , we used mice transgenic for the human mapt gene .
the transgene used is a 201  kb fragment of human genomic dna that contains the entire mapt gene and some flanking sequence .
the mice produce all six isoforms of the human tau protein and are null for the mouse mapt gene .
we injected the gastrocnemius muscles with either e5.3 or the control oligonucleotide or no injection .
histological analysis showed that these injections caused minimal tissue damage ( supplementary figure s11 ) .
we found that muscles ( left ) injected with e5.3 showed significantly less mapt mrna compared to noninjected muscles .
noninjected muscles had comparable levels of mrna and protein when compared to the control oligonucleotide ( figure 5a , b ) . e5.3
reduced tau protein from 18.1    3.6 to 5.3    0.85 pg tau/g protein ( p = 0.02 , figure 5c , d ) .
these results are comparable to those observed in neuroblastoma cell lines ( figure 3 ) .
the work presented here demonstrates the feasibility of reducing human tau protein levels using modified oligonucleotides complementary to mapt expressed mrna sequences .
exon boundaries , presumably by blocking access of splicing factors to the targeted exon . other strategies
included targeting intronic branchpoint sequences , polypyrimidine track sequences , exonic splicing enhancers , and intronic enhancer sequences were less successful .
exon skipping of exon 1 generates an mrna lacking an atg transcription start site and thus preventing normal translation .
exon skipping of either exons 1 , 5 , or 7 will result in changing the open reading frame of the mrna leading to a premature stop codon and possible nonsense - mediated decay .
the exon - skipping strategy used here to reduce protein levels differs from the exon skipping work with the dmd gene . in the latter case ,
exon skipping is used to by - pass exons with premature stop codon dmd mutations , but retain an open reading frame to produce a functional protein .
one of the most potent oligonucleotide was e1.4 , a morpholino complementary to the splice donor site of exon1 .
this oligonucleotide induces exon 1 skipping and results in mrna lacking exon 1 , with exon 0 spliced directly to exon 4 .
the transcript produced in the presence of this oligonucleotide lacks the translation start atg sequence , and the reading frame is changed leading to a premature stop codon at amino acid 20 encoded by exon 4 and another stop codon at amino acid 151 encoded by exon 9 .
e1.4 reduces mapt mrna levels by 5055 percent and tau protein synthesized ( figure 2 ) .
the reduced mrna levels , suggest that the introduction of a premature stop codon may lead to nonsense mediated decay .
the end result is tau protein is reduced to 2050% of normal expression in the two cell lines . although it is possible that an alternative atg site could be used , we did not detect a shorter than normal form of tau by immunoblotting ( figure 2 ) , though protein produced at a very low level would possibly not be detected .
oligonucleotides targeting exons 5 and 7 also effectively reduced both mrna and protein levels ( figure 3 and supplementary figure s9 ) .
again , the most effective morpholino spanned the 3 end of the exon and the beginning of the next intron .
the most effective morpholino was e5.3 , which reduced tau protein to 42% of normal levels in sh - sy5y cells ( figure 3d ) .
the resulting mrna contained exon 4 spliced to exon 7 using the normal spice sites for these two exons ( supplementary figure s8 ) , and no cryptic splice site mrna species were detected .
mutations that change isoform ratios cause ftld - t , either by increasing or decreasing inclusion of exon 10 .
the cell lines used here make mrna both lacking exon 10 ( 3r tau ) and with exon 10 ( 4r tau ) ( supplementary table s1 ) .
the most effective oligonucleotides tested here ( e1.4 and e5.3 ) reduced expression of both 3r and 4r mrna ( figure 4 ) showing that the 3r/4r isoform ratio is not significantly altered by these two morpholinos .
interestingly , while oligonucleotides targeting exon 4 splice sites did not significantly reduce total mapt mrna levels ( supplementary figure s3 ) , the amount of 4r mrna was reduced ( figure 4f ) .
exclusion of exon 4 does not alter the reading frame of mapt , but could change the kinetics of overall mapt rna processing such that the downstream alternatively spliced exon 10 is less likely to be included .
other more direct mechanisms are also possible . in the neuroblastoma cell lines used here ,
this result in surprising because the sequences known to regulate exon 10 splicing are located close to or within exon 10 ( refs .
also , kalbfuss et al . observed that antisense oligonucleotides complementary to exon 10 splice sites reduced exon 10 inclusion in mrna from a minigene containing mapt exon 10 , and in endogenous tau mrna from pc12 cells . in both these systems ,
the minigene contained mutations that increased exon 10 inclusion , and rodent cells ( pc12 ) normally produce predominantly 4r ( exon 10 + ) isoforms . in the work reported here
using human neuroblastoma cell lines , 3r tau ( exon 10- ) is the predominant mrna species .
thus the regulation of exon 10 inclusion in cells making mostly 4r tau mrna may differ from the regulation in cells that mostly exclude exon 10 , and the sequences and splicing factors that act when exon 10 inclusion is favored may not function the same when exon 10 is primarily excluded . as a proof of concept , we show that the most potent oligonucleotide used here can reduce human mapt expression in vivo in muscle .
reduction of tau levels in the brain is a potential therapeutic for multiple tauopathies including ad .
certainly the fact that mice lacking mapt are relatively normal argues that reduction of mapt expression in an adult could be well tolerated .
when antisense oligonucleotides against mapt sequences are introduced into mouse brain , mapt mrna is reduced as is mouse tau protein , and susceptibility to induced seizures is decreased .
this work also shows the feasibility of reducing brain tau as a therapeutic for at least seizures if not ad .
the primary barrier to using morpholinos for treatment is achieving adequate levels of modified oligonucleotides in the brain .
several strategies for getting oligonucleotides to cross the blood brain barrier have been described , though these approaches have not yet been tried in man .
other approaches include infusion of oligonucleotides into the cerebrospinal fluid or by use of implanted pumps .
while oligonucleotide - based therapies are attractive in that any expressed sequence could be targeted , delivery of adequate levels to oligonucleotides to the target tissue needs to be addressed .
cells . human neuroblastoma cell lines , sh - sy5y and imr32 , were used in this study .
imr32 cells ( atcc # ccl-127 ) were maintained in eagle 's minimum essential medium ( atcc # 30 - 2003 ) containing 10% fetal bovine serum ( gibco ) and 1% penicillin - streptomycin ( gibco ) .
lee ( center for neurodegenerative research , university of pennsylvania ) , were maintained in 1:1 mix of ham 's f12 and dulbecco 's modified eagle 's medium , supplemented with 10% fetal bovine serum , 1% penicillin - streptomycin , and 1% nonessential amino acids ( gibco ) .
both cell lines were maintained in a humidified incubator with 5% co2 . designing antisense morpholino oligonucleotides .
the morpholino oligonucleotides used were designed to hybridize to exon splice sites , branchpoint sequences , and regulatory sequences in or close to mapt exons .
splice site morpholino oligonucleotides were picked based on splice - site predictions from sroogle and on previously determined intron  exon boundaries . wherever possible , we used a  sliding window design  to generate a panel of antisense oligonucleotides for blocking the same splice site .
exon 10 oligonucleotides were designed to hybridize to regulatory sequences in the exon and in intronic sequences that immediately follow this exon .
blast analyses were performed for each morpholino oligonucleotide sequence to avoid off - target hybridization .
nucleofection with no oligonucleotide added and a negative control morpholino oligonucleotide ( gene - tools ) that targets mutated -globin transcripts .
this control oligonucleotide did not affect tau levels in either imr32 or sh - sy5y cells or in transgenic mice ( figures 2 and 5 and supplementary figure s11 , respectively ) .
morpholino oligos were introduced in neuroblastoma cells with the amaxa nucleofector - ii device ( lonza , walkersville , md ) . for initial testing of morpholino efficacy ,
nucleofector solution sf was selected after preliminary experiments , based on high viability and efficient transfection .
for nucleofection of neuroblastoma cell lines in six - well plates , amaxa recommended kits v and l were used for introducing antisense morpholinos in sh - sy5y and imr32 cells respectively . on the day of nucleofection
, 6080% confluent cultures were trypsinized , and cells plated in each well of six - well plates . at 24 hours postnucleofection ,
sample of nucleofected cells were analyzed by flow cytometry to ascertain % of cells positive for carboxyfluorescein - tag on the morpholino oligonucleotides . in all cases ,
cells were harvested at day-6 postnucleofection for analysis of rna and protein . at day 6
, there was no significant difference in viability of mock and morpholino - treated cultures using an 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay ( data not shown ) .
lysis and further processing was performed using the illustra tripleprep kit ( ge healthcare , fairfield , ct ) per manufacturer 's instructions .
lysates were homogenized by passing five times though a 22g - needle attached to 1cc syringe .
homogenates were stored at 80 c till extraction of rna and protein , performed as per manufacturer 's instructions .
briefly , antisense - treated cells were lysed in kit - supplied buffer , supplemented with 1% 2-mercaptoethanol . after removing gdna
, acetone was added to precipitate rna , subsequently bound to silica columns and later collected in elution buffer .
kit - supplied precipitation solution was used to pellet the proteins , subsequently solubilized in 2d - dige buffer ( 7  mol / l urea , 2  mol / l thiourea , 4% ( v / v ) 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate ( chaps ) , 30 mmol / l tris - hcl , ph 8.5 ) . for immunoblots ,
tau levels were analyzed in cytoplasmic lysates prepared in the following buffer : 125 mmol / l tris - hcl ( ph 6.8 ) , 200 mmol / l nacl , 2.5 mmol / l mgso4 , 0.1% triton - x , and 0.5% np-40 .
immediately before addition to cell pellets , lysis buffer was supplemented with protease inhibitor cocktail ( roche diagnostics , indianapolis , in ) .
tau isoforms expressed in neuroblastoma lysates were identified by running recombinant standard ( sigma , st louis , mo # t7951 ) .
rt - pcr experiment design and reporting of results is based on minimum information for publication of quantitative real - time pcr experiments ( miqe ) guidelines .
the concentration and purity of isolated rna was determined using the nanodrop 2000 spectrophotometer ( thermo scientific , waltham , ma ) .
representative rna samples from different preparations were also assessed for integrity of isolated rna using an agilent 2100 bioanalyzer .
rna template in each experimental paradigm was reverse transcribed to cdna using first - strand cdna synthesis kit ( ge healthcare ) or the high capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) .
changes in mapt expression were detected quantitative real - time pcr was performed using a 7900ht fast real time pcr system ( applied biosystems ) with taqman fast universal pcr master mix and applied biosystems taqman gene expression assay mix : i d # hs00902194_m1 .
this probe binds the junction of mapt exons 1213 ; constitutive exons present in all isoforms and therefore this assay helps determine total levels of all mapt transcripts . for 3r isoform detection , assay hs00902192_m1
was used with a probe that binds to the exon 9  exon 11 junction fragment . for 4r isoform detection , assay hs00902312_m1
fold changes were calculated by the 2 t compared to mock - transfected (  no oligo  ) cultures . for this control ,
ct equals zero and 2 equals one , so that the fold change in gene expression relative to the mock - transfected control equals one by definition .
data were normalized to endogenous reference genes ( 18s rrna , -actin , gapdh ) and plotted as % change relative to the mock or  no oligonucleotide  control in figures .
did exclude targeted exons , cdna from treated cells was amplified with pcr primers complementary to mapt sequences outside the skipped exon ( supplementary table s2 ) .
subsequent analyses revealed shorter transcript fragments in oligo - treated cultures , when compared to mock - cultures .
parameters for pcr amplification were : 95 c/15 minutes ; 30 cycles of 94 c/30 seconds , 60 c/30 seconds , 72 c/1 minute ; 72 c/10 minutes .
total cellular protein extracted was quantified with qubit assay ( invitrogen , carlsbad , ca ) .
cell lysates were dephosphorylated using -phosphatase ( new england biolabs , ipswich , ma ) as previously described .
primary antibody for detecting differences in tau levels post - antisense treatment was purchased from santa cruz biotechnology ( dallas , tx ) ( sc-21796 , at 1:1,000 ) . for detection , secondary antibody # na931v ( ge healthcare )
both antibodies were diluted in 5% nonfat milk in 1 phosphate - buffered saline ( gibco , carlsbad , ca ) supplemented with 0.1% tween-20 .
b - actin ( cell signaling , beverly , ma ) was used as loading control .
morpholino - induced changes in tau protein were quantified with elisa ( invitrogen ) per manufacturer 's instructions .
the intramuscular ( i.m . ) injections were used a proxy model to explore whether morpholino oligonucleotides that effectively reduced tau protein levels in vitro would work in vivo . the transgenic mouse used
was described previously ( http://jaxmice.jax.org/strain/017459.html ) and harbors a point mutation in intron 10 ( e10 + 14 ) that causes frontotemporal dementia .
control group included animals injected with negative control morpholino , saline , and with vehicle 
kollliphor p407 ( formerly called pluronic f-127 ) , a kind gift from basf , white plains , ny .
p407 is a nonionic polymer previously used successfully in dmd mouse models to facilitate intracellular delivery of antisense oligonucleotides following i.m .
animals were injected with 20 g/l morpholino and p407 at a final concentration of 40 g/l , diluted in saline , in a total volume of 30 l per muscle .
samples included noninjected controls , gastrocnemius muscles from : saline - injected , vivo - morpholino injected , morpholino + p407 coinjected tissue .
stained slides were visualized using a leica dm4000b microscope and photographed with a leica dfc 450 camera . statistical analysis . in case of rt - pcr ( taqman ) and elisa experiments , changes to tau levels were compared to  mock  , or no - oligo control . statistical evaluation
treatment with exon 0-specific antisense oligo did not affect mapt transcript levels 

 figure s2 .
exon 1 is omitted after treatment with morpholino oligos targeting splice sites to this exon . 


effect of varying concentrations of e5.3 antisense oligo on total mapt transcripts in imr32 cells . 


exon 5 is skipped after treatment with morpholino oligos targeting splice sites to this exon . 


primers used to sequence mapt in cdna isolated from morpholino - treated cultures or corresponding controls .
treatment with exon 0-specific antisense oligo did not affect mapt transcript levels click here for additional data file .
click here for additional data file . targeting splice sites to exon 4 with morpholinos did not influence tau levels .
exon 1 is omitted after treatment with morpholino oligos targeting splice sites to this exon .
effect of varying concentrations of e5.3 antisense oligo on total mapt transcripts in imr32 cells .
exon 5 is skipped after treatment with morpholino oligos targeting splice sites to this exon .
histological analyses of injected muscles comparing morpholino oligonucleotides of different chemistries click here for additional data file .
primers used to sequence mapt in cdna isolated from morpholino - treated cultures or corresponding controls .
 in alzheimer 's disease , progressive supranuclear palsy , and a number of other neurodegenerative diseases , the microtubule associated protein tau aggregates to form intracellular neurofibrillary tangles and glial tangles , abnormal structures that are part of disease pathogenesis . 
 disorders with aggregated tau are called tauopathies . 
 presently , there are no disease - modifying treatments for this disease class . 
 tau is encoded by the mapt gene . 
 we propose that reducing mapt expression and thus the amount of tau protein made could prevent aggregation , and potentially be an approach to treat tauopathies . 
 we tested 31 morpholinos , complementary to the sense strand of the mapt gene to identify oligonucleotides that can downregulate mapt expression and reduce the amount of tau protein produced . 
 oligonucleotides were tested in human neuroblastoma cell lines sh - sy5y and imr32 . 
 we identified several morpholinos that reduced mapt mrna expression up to 50% and tau protein levels up to ~80% . 
 the two most potent oligonucleotides spanned the 3 boundary of exons 1 and 5 , masking the 5-splice sites of these exons . both morpholinos induced skipping of the targeted exons . 
 these in vitro findings were confirmed in mice transgenic for the entire human mapt gene and that express human tau protein . 
 these studies demonstrate the feasibility of using modified oligonucleotides to alter tau expression . 
Introduction
Results
Effects of oligonucleotides on total MAPT mRNA levels and total tau protein
Effects of morpholino oligonucleotides on inclusion of exon 10
None
Discussion
Materials and methods
Supplementary Material