Patent Application: US-201213644363-A

Abstract:
certain disclosed oligomers induce exon skipping during processing of myostatin pre - mrna . the oligomers may be in a vector or encoded by the vector . the vector is used for inducing exon skipping during processing of myostatin pre - mrna . a therapeutically effective amount of the oligomer may be administered to a subject patient such that exon skipping during processing of myostatin pre - mrna is induced . the administration to a subject may be used in order to increase or maintain muscle mass , or slowing degeneration of muscle mass in the subject . the administration to a subject may ameliorate muscle wasting conditions , such as muscular dystrophy . examples of such muscular dystrophies which may be so treated include becker &# 39 ; s muscular dystrophy , congenital muscular dystrophy , duchenne muscular dystrophy , distal muscular dystrophy , emery - dreifuss muscular dystrophy , facioscapulohumeral muscular dystrophy , limb - girdle muscular dystrophy , myotonic muscular dystrophy , and oculopharyngeal muscular dystrophy

Description:
the present invention will now be described in detail , by way of example only , with reference to the accompanying figures in which : fig1 : bioinformatics analysis , design , and evaluation in c2c12 muscle cells of specific aos predicted to induce skipping of myostatin exon 2 . ( a ) results from three algorithms used to identify ese sequences for designing of exon skipping aos targeting exon 2 of the mouse myostatin gene . the ese finder analysis shows the location and values above threshold for sr protein - binding motifs , sf2 / asf , sf2 / asf ( brca 1 ), sc35 , srp40 , and srp55 which are shown as vertical bars above the sequence of exon 2 . the rescue ese analysis shows the position of possible exonic splicing enhancer sites by black horizontal lines parallel to the sequence of exon 2 . the pesx analysis shows the location of eses as light gray horizontal lines , and exon splicing silencers ( esss ) as dark gray horizontal lines . the bold horizontal laddered black lines represent the sequence of the 20 - mer 2 &# 39 ; omepss which were obtained after aligning the outputs from the three algorithms . ( b ) comparison of efficacy of different 2 &# 39 ; omeps oligomers to induce skipping of exon 2 in myostatin mrna from c2c12 cell cultures . rt - pcr was performed on 1 μg mrna from c2c12 cells treated with 12 different 2 &# 39 ; omeps oligomers at 250 nmol / l . transfections were performed in duplicates and the nested rt - pcr products were loaded on 1 . 2 % agarose gel as follow : tracks 1 and 2 : oligomer a1 ; tracks 3 and 4 : oligomer a2 ; tracks 5 and 6 : oligomer a3 ; tracks 9 and 10 : oligomer b1 ; tracks 11 and 12 : oligomer b2 ; tracks 13 and 14 : oligomer b3 ; tracks 15 and 16 : oligomer c1 ; tracks 17 and 18 : oligomer c2 ; tracks 19 and 20 : oligomer c3 ; tracks 23 and 24 : oligomer d1 ; tracks 25 and 26 : oligomer d2 ; tracks 27 and 28 : oligomer d3 ; tracks 7 , 8 , 21 and 22 : controls with transfection reagent lipofectamine 2000 alone , but no ao : size marker used is hyper ladder iv . 2 &# 39 ; omeps , 2 &# 39 ; o - methyl phosphorothioate rna ; ao , antisense oligonucleotide ; bp , base pairs ; ese , exonic splicing enhancer ; rt - pcr , reverse transcriptase - pcr . fig2 : antisense - induced myostatin exon 2 skipping with 2 &# 39 ; omeps oligomers leads to an increase in c2c12 cell proliferation . c2c12 cells were treated with a range of 2 &# 39 ; omeps oligomers along with lipofectamine 2000 ( lf2000 ), and assayed 24 hours later for cell proliferation by lactic dehydrogenase assay . treatment with 2 &# 39 ; omeps oligomers a3 , b3 , and d3 resulted in significant increases in the number of cells after 24 hours compared to the cultures treated with only transfection reagent lf2000 but no ao . c3 did not induce a substantial change . ( t - test analysis , n = 6 ; * p & lt ; 0 . 05 ; ** p & lt ; 0 . 01 ). 2 &# 39 ; omeps , 2 &# 39 ; o - methyl phosphorothioate rna . fig3 : exon skipping in mouse following intramuscular injection of 2 &# 39 ; omeps oligomers targeting myostatin exon 2 . oligomers a2 and b3 ( 3 nmol ) were administered by a single intramuscular injection into the tibialis anterior ( ta ) muscles of mice . two and four weeks later , muscles were recovered , and rna extracted and analyzed for the presence of myostatin exon 2 skipping by rt - pcr . agarose ethidium bromide gel electrophoresis is shown for the products of rt - pcr analysis : the upper and lower bands correspond to the normal exon 1 , 2 , and 3 product ( 532 bp ) and the exon 2 skipped product ( 158 bp ), respectively which were verified by sequencing ( data not shown ). the faint shadow band of intermediate migration in some tracks was found upon sequencing to correspond to a product containing a partial sequence of exon 2 due to a cryptic 3 ′ splice site 296 nt downstream of the correct one . tracks 1 and 2 : 14 days control ; tracks 3 - 5 : 14 days a2 - treated ; tracks 6 - 8 : 28 days a2 - treated ; tracks 9 - 11 ; 14 days b3 - treated ; tracks 12 - 14 : 28 days b3 - treated ; tracks 15 and 16 : 28 days control . densitometric evaluation of the skipped and unskipped bands showed that after 14 days , a2 gave 25 . 6 % and b3 54 . 6 % skipping , and after 28 days , a2 gave 48 . 6 % and b3 24 . 5 % skipping . 2 &# 39 ; omeps , 2 &# 39 ; o - methyl phosphorothioate rna ; bps , base pairs ; nt , nucleotides ; rt - pcr , reverse transcriptase - pcr . fig4 : myostatin exon 2 skipping in c2c12 cell culture following treatment with a range of leashed - pmo lipoplexes . c2c12 cell cultures treated with a range of leashed pmos in lipoplex form with lf2000 exhibited skipping of exon 2 in myostatin mrna . rt - pcr was performed on 1 μg mrna from c2c12 cells treated with 250 nmol / l pmos ( designed on the basis of the most effective 2 &# 39 ; omeps sequences : a3 , b3 , c3 , and d3 ) over a period of 24 hours . transfections were performed in triplicate and rt - pcr products were loaded on 1 . 2 % agarose gel as follows : tracks 1 - 3 : pmo - a ; tracks 4 - 6 : pmo - b ; tracks 7 - 9 : pmo c ; tracks 10 - 12 : pmo - d ; tracks 13 - 15 : lf2000 - treated control ; bps , base pairs ; lp2000 , lipofectamine 2000 ; pmo , phosphorodiamidate morpholino oligomers ; rt - pcr , reverse transcriptase - pcr . fig5 : systemic injection of pmo conjugated to octa - guanidine dendrimer ( vivo - pmo ) results in myostatin exon skipping associated with a significant increase in muscle mass and myofiber size . mice were treated with 6 mg / kg of vivo - pmo - d3 by five weekly intravenous injections , and muscles harvested for rna extraction and immunohistology 10 days later . ( a ) weight of soleus and edl muscle after treatment . weights of soleus muscles were significantly increased ( t - test , p & lt ; 0 . 034 ; n = 6 ) whereas weights of edl muscles showed no significant change . ( b ) rt - pcr was carried out on 1 μg rna from soleus and edl muscles and products resolved on a 1 . 2 % agarose gel . track 1 : vivo - pmo treated soleus ; track 2 : control soleus ; track 3 : vivo - pmo treated edl ; track 4 : control edl . ( c ) distribution of myofiber sizes ( csa ) in vivo - pmo treated ( black bars ) and control ( open bars ) soleus muscles . ( d ) representative dystrophin immunohistology indicating increased myofibre csa in vivo - pmo treated compared to control soleus muscle cryosections . bar = 500 μm . csa , cross - sectional area ; edl , extensor digitorum longus ; pmo , phosphorodiamidate morpholino oligomers ; rt - pcr , reverse transcriptase - pcr . as stated above , the inventors have adopted the approach of using aos with different chemistries , so enhancing their half - lives relative to rnai molecules , to investigate the outcome of myostatin knockdown by exon skipping . skipping of exon 2 ( 374 nucleotides ) of myostatin was predicted to lead to an out - of - phase splicing of exons 1 and 3 , and knockdown of myostatin due to truncation of the open reading frame and nonsense - mediated mrna decay . the data present below constitute a proof - of principle that oligonucleotide - mediated antisense exon skipping leads to a physiologically significant myostatin knockdown in vitro and in vivo . this type of antisense treatment could thus potentially form part of an effective strategy to improve various muscle - wasting conditions , and along with dystrophin rescue or augmentation , to treat duchenne muscular dystrophy . three different bioinformatics algorithms namely ese finder , pesx , and rescue ese were used to design antisense reagents . results from the three algorithms were merged to define ese sites and used to identify the regions of the myostatin exon 2 , which are expected to be optimal targets for exon skipping antisense reagents . a set of 12 antisense reagents of 2 &# 39 ; o - methyl rna ( 2 &# 39 ; omeps ) chemistry were designed to target four different ese - rich regions of exon 2 of myostatin ( fig1 a ). the 12 2 &# 39 ; omeps oligomers tested were obtained from eurogentec ( sa , seraing , belgium ). the sequences of the 2 &# 39 ; omeps are as follows : pmos were designed based on the 2 &# 39 ; omeps sequences . a total of four pmos were tested and were obtained from gene tools ( philomath , oreg .). pmo sequences are as follows : pmos conjugated to octa - guanidine dendrimers ( so - called vivo - pmos ) were purchased from gene tools . cell culture and transfection of c2c12 cells with the designed antisense reagents . c2c12 mouse myoblasts were maintained in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( sigma - aldrich , poole , uk ) containing 10 % fetal calf serum ( sigma - aldrich ), 4 mmol / l 1 - glutamine , 100 u / ml penicillin and 100 μg / ml streptomycin at 37 ° c . and 8 % co 2 . cells were split every 24 hours to prevent differentiation . cells were detached by incubating them with 0 . 15 % trypsin - phosphate - buffered saline for 1 minute at 37 ° c ., and seeded at a density of 1 . 5 × 105 cells / well of a 6 - well plate . the antisense reagents of 2 &# 39 ; omeps chemistry were transfected at 250 nmol / l into c2c12 cells using lipofectamine 2000 ( invitrogen , paisley , uk ). controls contained lipofectamine 2000 but no antisense reagent . pmos were leashed to complementary stretches of negatively charged dna ( obtained from mwg , ebersberg , germany ) for efficient in vitro delivery , 24 using lipofectamine 2000 as transfection reagent . all transfections were performed in dulbecco &# 39 ; s modified eagle &# 39 ; s medium containing 2 mmol / l glutamine ( without serum and antibiotics ) and after 3 - 4 hours of transfection , the medium was replaced with full growth medium containing serum as well as antibiotics . the transfections were performed in duplicate and the experiment repeated twice . for in vitro experiments , 24 hours after transfection , rna was extracted from each well using qiashredder / rneasy extraction kit ( qiagen , crawley , uk ). for in vivo experiments , rna was extracted from blocks using trizol reagent ( invitrogen , scotland , uk ). one microgram of rna was reverse transcribed and resulting complementary dna amplified using specific primers obtained from mwg , using the genescript kit ( genesys , camberley , uk ). one micro litre of pcr products obtained was used as a template for nested pcr . sequences of the primers and details of the pcr protocols used are available on request . the products from nested pcr were separated on 1 . 2 % agarose gel in tris - borate / edta buffer and hyper ladder iv ( bioline , london , uk ) was used as the marker . densitometric analysis of the agarose gels was carried out using gene tools 3 . 05 ( syngene , cambridge , uk ) and percentage skipping expressed as amount of skipped product seen relative to total pcr products detected . a proliferation assay using cell titer 96 aqueous one solution cell proliferation assay ( promega , madison , wis .) was performed , as reported by cory et al ., 75 on cells transfected with different 2 &# 39 ; omeps . briefly , 24 hours after seeding , the growth media was replaced with serum - free media and cells incubated at 37 ° c . after 24 hours of subjecting cells to serum - free media , 15 μl of assay reagent was added to 75 μl cells in a 96 - well plate . plates were read at 490 nm . statistical analysis on the data from the proliferation assay was performed using the individual t - test . for all the in vivo experiments , animals ( mf1 or c57b110 ) were bought from harlan ( blacktown , uk ) and in - house maintained , and in vivo experimentation conducted under statutory home office recommendation , regulatory , ethical and licensing procedures and under the animals ( scientific procedures ) act 1986 ( project licence ppl 70 / 7008 ). for intramuscular delivery , mice were anaesthetized and injected with 3 nmol of 2 &# 39 ; omeps ( in 25 μl normal saline ) into each of the ta muscles . control animals were injected with normal saline . whole body weights were measured weekly . tas of treated and control mice were excised postmortem after 2 weeks ( n = 4 ) and 4 weeks ( n = 4 ). weights were measured and the muscles frozen in iso - pentane cooled with liquid nitrogen . for the systemic administration , c57bl10 mice were injected intravenously with 6 mg / kg of vivo - pmo - d ( gene tools ) diluted in 200 μl of normal saline , every week for 5 weeks . weights were measured weekly and various muscles from treated and control mice were harvested 10 days after the last injection . cryosectioning was performed at 10 levels through the muscle . hematoxylin and eosin staining was used to estimate the muscles size . for the estimation of fibre size and distribution , laminin staining was performed . laminin antibody from sigma - aldrich ( dorset , uk ) was used as primary antibody , with biotinylated anti - rabbit immunoglobulin g ( dako , glostrup , denmark ) as secondary antibody . finally sections were stained with dab ( vector laboratories , burlingame , calif .) and slides mounted in dpx ( vwr international , poole , england ) after appropriate washings . immunostaining was also carried out with dystrophin antibody . for this , h12 polyclonal rabbit antibody was used as primary antibody , and alexa fluor goat anti - rabbit 568 ( fluorescein isothiocyanate ) ( invitrogen , paisley , oreg .) was used as secondary antibody . slides were mounted in vectashield mounting medium with dapi ( vector laboratories ) after appropriate washings with phosphate - buffered saline - tween . csa of muscle fibres was measured using sigmascan pro 5 . 0 . 0 ( systat software , london , uk ). bioinformatics analysis and design of specific aos predicted to induce skipping of myostatin exon 2 . bioinformatics analysis of exon 2 of myostatin was performed using three bioinformatics tools , ese finder , 27 , 28 pesx , 29 , 30 and rescue ese , 31 to identify and locate eses and exonic splicing suppressor or silencer motifs . the output of these algorithms is displayed in fig1 a . a series of overlapping aos were designed and synthesized as 2 &# 39 ; omepss and pmos to span sequences where clusters of eses which were predicted by one or more of the programs coincide . high levels of myostatin exon 2 skipping in c2c12 cell culture following treatment with a range of aos . in order to verify the efficiency of these ao target sequences , c2c12 muscle cell cultures were transfected with the 2 &# 39 ; omeps oligomers , and nested reverse transcriptase - pcr ( rt - pcr ) for skipping of myostatin performed on the rna extracted from transfected and control cells . a representative horizontal agarose gel electrophoresis separation of products obtained is shown in fig1 b . the level of skipping produced by each ao at 250 nmol / l was determined semiquantitatively using densitometric analysis . 31 all of the designed 2 &# 39 ; omepss were observed to induce myostatin exon 2 skipping in c2c 12 cultures but at various levels of relative efficiency . a2 and a3 induced almost 100 % skipping ; b3 ( 74 %), c3 ( 41 %), and d3 ( 48 %) also induced a considerable level of skipping . the nature of putative antisense - induced pcr exon1 - exon3 splicing product was confirmed by sequencing the products . antisense - induced myostatin exon 2 skipping and myostatin knockdown leads to an increase in c2c12 cell proliferation . in order to verify that ao - mediated myostatin exon 2 skipping and knockdown , lead to a significant biological response , the autocrine activity of myostatin on c2c12 cell proliferation was evaluated following treatment of cultures with 2 &# 39 ; omepss targeting myostatin exon 2 . the cell proliferation assay was based on determination of lactic dehydrogenase activity of metabolically active cells . the results of the proliferation assay clearly showed a remarkable difference in cell proliferation in c2c12 cells treated with myostatin exon 2 aos compared to mock - transfected control cells ( fig2 ). statistical analysis of the data using individual paired t - tests showed that oligomers a3 ( p = 0 . 0031 ), b3 ( p = 0 . 0055 ) and d3 ( p = 0 . 0115 ) induced a significant increase in cell proliferation , as compared to mock transfected control cells . oligomer c3 ( p = 0 . 0534 ) did not produce a statistically significant change . demonstration of exon skipping in mouse following intramuscular injection of 2 &# 39 ; omeps oligomers targeting myostatin exon 2 . on the basis of rt - pcr results obtained from the in - vitro studies , two 2 &# 39 ; omeps oligomers ( a2 and b3 ) were selected to evaluate their ability to induce efficient exon skipping in vivo . the 2 &# 39 ; omeps oligomers ( 3 nmol ) were administered by intramuscular injection into tibialis anterior ( ta ) muscles of mice . two and four weeks after the injections , muscles were recovered , weighed , rna extracted and analyzed for the presence of myostatin exon 2 skipping by rt - pcr . both reagents ( a2 and b3 ) induced significant level of myostatin exon 2 skipping at either the 2 weeks and 4 weeks time points after a single 2 &# 39 ; omeps oligomer administration ( fig3 ). densitometric quantification of full - length and skipped product bands from the rt - pcr analyses of rna was performed to detect which of the two 2 &# 39 ; omepss tested was the more efficient in vivo . oligomer a2 gave 25 . 6 % skipping , and b3 gave 54 . 6 % skipping at the 2 weeks time point . however , after 4 weeks , a2 gave 48 . 6 % skipping whereas b3 gave 24 . 5 % skipping . although the skipping of myostatin exon 2 was evident , the effect was not sufficient to see a significant change in ta muscle mass ( data not shown ). from previous work on exon skipping for dystrophin , it is well established that the intramuscular injections of naked unconjugated aos in undamaged muscles are not very efficient . 25 high levels of myostatin exon 2 skipping in c2c12 cell culture following treatment with a range of pmos designed on the basis of 2 &# 39 ; omeps data . the animal studies above established that exon skipping of the myostatin gene observed after intramuscular injection of 2 &# 39 ; omeps aos was insufficient to induce change in ta mass . the pmo chemistry has been demonstrated to have very high efficiency in vivo . 33 therefore , pmos were designed on the basis of most efficient 2 &# 39 ; omeps aos ( a3 , b3 , c3 , and d3 ) and initially tested in vitro . pmos are uncharged chemicals and do not directly interact with the polycationic transfection reagent lipofectamine 2000 . in order to enable reasonable transfection efficiency in c2c 12 cells , pmos were hybridized to complementary so - called leash oligonucleotides of natural negatively charged dna chemistry as previously described . 23 , 34 , 35 nested rt - pcr analysis of mrna harvested from c2c12 cells treated with leashed - pmo lipoplexes demonstrated that exon skipping was induced by all the pmos accurate skipping of the targeted exon by both ao chemistries tested here . systemic injection of pmos conjugated to octa - guanidine dendrimer resulted in myostatin exon skipping associated with a significant increase in muscle mass and myofibre size . the conjugation of pmo with octa - guanidine dendrimer ( so - called vivo - pmos ) significantly increases the delivery and efficiency of pmo directed against exon 23 of dystrophin compared to unmodified pmo . 25 therefore , a vivo - pmo based on the sequence of the previously tested 2 &# 39 ; omeps oligomer , d3 , was produced to evaluate systemic intravascular treatment regimes . mice were treated with 6 mg / kg of vivo - pmo - d3 by five weekly intravenous injections , and whole body weight and the mass of ta , soleus , and extensor digitorum longus ( edl ) muscles were recorded 10 days after the last injection . among the muscles analyzed in the treated animals the soleus showed a statistically significant change in mass ( p = 0 . 034 ) ( fig5 a ). in accordance with this , high levels of exon skipping of myostatin exon 2 was demonstrated at the transcript level in soleus ( 79 %), whereas a very low level of skipping was observed in edl muscle ( 9 %) ( fig5 b ). importantly , the cross - sectional area ( csa ) of soleus muscle fibres in treated animals significantly increased ( p & lt ; 0 . 0001 ; mean csa were 254 ± 5 μm2 for control and 333 ± 3 μm2 for pmo - treated animals ( n = 6 ) with a significant shift on the distribution of csa ( x2 = 38 . 34 ; df = 12 ) ( fig5 c , d ). no change was observed in the csa of edl muscle . although targeting donor splice site , acceptor splice sites and branch point sequences has successfully led to exon exclusion including dmd exon skipping 36 - 38 , some studies have proved that targeting splice sites does not always induce exon skipping and therefore exclusion of an exon from the pre - mrna 39 these contain some consensus sequences common to many other genes ; therefore there lies a possible risk of disrupting the splicing of non - specific genes 40 . exon splicing enhancers ( eses ) motifs form the binding sites for sr - protein rna domains and thus help the splicing machinery in exon recognition 41 . it has been shown that intraexonic point mutations usually lead to mrna level of exon skipping instead of misense or no change in the amino acids 42 . as sr protein binding to eses is very crucial for exon exclusion , blocking the eses with antisense oligonucleotides ( aos ) would be expected to result in exon skipping . different software like rescue ese 43 , 44 , esefinder 45 and pesx 46 have been widely used to predict possible ese sites for different sr domains in order to assist in designing aos 40 , 47 , 48 . different oligonucleotide sequences of two different chemistries to target myostatin exon 2 were designed using these available online tools which showed a promising level of exon skipping . 2 &# 39 ; omeps chemistry was used for the preliminary tissue culture studies because of the advantage of cheap and easy synthesis over some other the pmo chemistry 49 , 50 . rna from all the c2c12 cells transfected with twelve different 2 &# 39 ; omeps ao sequences showed skipped myostatin exon 2 as demonstrated by rt - pcr along with the full length product . as myostatin has been established to be a negative regulator of muscle mass growth and differentiation 51 - 53 , a decrease in its level is expected to result in enhanced proliferative capacity of muscle cells 54 , 55 . therefore , a colorimetric proliferation assay based on the principle of bioreduction of a tetrazolium compound by viable cells gives a quantitative measure of living cells present in a system 56 . on performing this assay on cells treated with four different aos ( one from each of the four sets based on rt pcr results ), it was observed that the cells treated with ao - a3 showed increased proliferation compared to control cells ( p & lt ; 0 . 01 ), treatment with aos b3 and d3 also showed an increased cell proliferation ( p & lt ; 0 . 05 ), whereas ao - c3 did not lead to a significant increase in level of proliferation compared to the control cells . pmo chemistry has high nuclease resistance 57 and it does not induce rnase h - mediated down - regulation of the mrna that it targets 58 . due to uncharged background , however , pmos cannot be delivered efficiently across cells using cationic liposomes and needs to be used at very high concentrations 59 , 60 . therefore , an anionic single - stranded nucleic acid molecule called ‘ leash ’ was annealed to the pmo in order to mediate complex formation of pmo with the cationic transfection reagent 61 . four different 20 - mer 2 &# 39 ; omeps ao sequences were used for design and synthesis of pmos . pmos were 30 - mers with an overlap of 10 bases between ao - 2 and ao - 3 of each of the 2 &# 39 ; omeps ao sets , a ( 1 , 2 , 3 ), b ( 1 , 2 , 3 ), c ( 1 , 2 , 3 ) and d ( 1 , 2 , 3 ). all the pmos linked to their respective leashes resulted in induction of exon 2 skipping of myostatin mrna and therefore showed the feasibility of the approach with two different ao chemistries . a luciferase reporter assay has been used to study the myostatin inhibition effect of myostatin propeptide as well as that of myostatin neutralizing antibody ja16 in terms of a decrease in smad binding to a tgf - β responsive elements called caga boxes 62 - 64 . a dose - response curve was prepared using different dilutions of recombinant mouse myostatin using human rhabdomyosarcoma cell line , a204 transfected with pgl3 -( caga ) 12 - luc . when supernatant from c2c12 cells treated with pmo - d was assayed on the a204 cells transfected with luciferase reporter containing smad - binding site ( caga ) and luminescence was recorded , there was found to be a significant decrease ( p = 0 . 011 ) in the relative light units ( rlu ) after 48 hours in case of supernatant from treated c2c12 cells compared to supernatant from control c2c12 cells . this indicates a decrease in the transcriptional activity of endogenous smad proteins which are crucial for tgf - β - mediated signal transduction 62 therefore , inhibition of myostatin by exon skipping results in reduced biological activity related to modulation of myostatin pathway . this study thus confirms the reported results for myostatin blockade using dominant negative actriib in human myoblasts 65 myostatin - neutralizing antibody , ja16 64 and myostatin propeptide 63 showing a decrease in smad2 transcriptional activity and thus antagonizing biological activity . when the mean rlu values from the reporter assay for pmo - d treated cells were plotted on the myostatin standard curve , there was found to be a decrease in myostatin concentration in treated samples relative to the control cells by 44 %. all these results were evident of skipping of myostatin exon 2 in vitro using two different chemistries along with modulation of proliferative capacity as well as alteration of myostatin pathway of ao - treated cells . it has been well established that myostatin is a negative regulator of skeletal muscle mass 66 and several approaches have been used to knockdown this factor to induce an increase in skeletal muscle growth . 67 the use of aos to induce exon skipping and thereby knockdown the expression of myostatin presents several advantages over the other currently used gene therapy approaches . firstly , there is no risk of uncontrolled insertion into the genome with aos as in case of virus - mediated approaches . 68 moreover , with an appropriate dosing regimen , exon skipping levels can be regulated and , if necessary the treatment can be interrupted . importantly aos have not been reported to produce any toxic effects or immune response so far in animal models as well as when used in clinical application . 69 here , the inventors show that aos of 2 &# 39 ; omeps chemistry , designed using bioinformatics algorithms , resulted in a substantial level of myostatin exon 2 skipping in vitro . myostatin being an inhibitor of myogenic differentiation , controls the proliferation of myoblasts . 70 therefore , myostatin knockdown is expected to increase the cell proliferation . the aos designed were biologically active and induced an increase in c2c12 cell proliferation . the efficacy of knockdown by exon skipping in vivo has proved to be more challenging to establish than in vitro . the efficiency of myostatin skipping was verified by injecting 2 &# 39 ; omeps intramuscularly . the intramuscular treatment of a single muscle induced exon skipping , but did not appear to affect myostatin activity . this is likely to be due to supply of biologically active myostatin to the injected muscle by the bloodstream . moreover , in a hypothetical clinical approach , the whole body should be treated . for these reasons , the inventors decided to administer the aos by systemic tail vein injection in further experiments . pmo chemistry was chosen for this experiment due to its better stability compared to the 2 &# 39 ; omeps and also because pmos have been reported to have a longer effect in vivo . 71 this is particularly important for knocking down proteins like myostatin , which do not have a long half - life like dystrophin . the pmo sequence used for the systemic administration study induced efficient skipping in vitro . it also maps in a region totally conserved between mouse and human myostatin paving the way to test the same pmo for clinical applications in humans . in order to achieve a reasonable effect in undamaged muscles , a pmo conjugated to a delivery moiety has to be used . 72 vivo - pmo is commercially available and has been reported to be effective in normal healthy mice . 25 by injecting vivo - pmo , a substantial increase in muscle size and the change of csa fibre distribution has been obtained , but only in the soleus muscle . the differential response in edl and soleus may be due in part to a greater amount of actriib being expressed on the surface of edl muscle , or because the intrinsic level of myostatin is greater in fast ( myosin type iib positive ) myofibres . 4 , 6 alternatively , it can be speculated that the dosing regimen used , which has been reported to be optimal for vivo - pmo for exon skipping of dystrophin gene , 25 does not achieve sufficient skipping of myostatin gene in edl . in the case of dystrophin skipping , the half - life of dystrophin protein and mrna is extremely long and therefore relatively smaller dosage of ao gives more sustained exon skipping . 73 however , in myostatin skipping , it is perhaps likely that more frequent redosing is required , to have a more sustained presence of aos . this may explain the transient and weak effect in terms of whole body weight change that was observed in vivo . interestingly , only soleus muscle showed a significant increase in weight and csa fibre distribution . this is in compliance with some previously published data showing that soleus is the most affected muscle following a systemic approach to knockdown myostatin . 4 the results represent a proof - of - principle that myostatin knockdown can be obtained by skipping an exon from the transcript by using aos . the 30 - mer pmo aos tested above ( mstn - a to mstn - d ) were designed to target the myostatin gene in mice . therefore , these mstn - a to mstn - d sequences correspond ( are complementary to ) to the genbank mouse myostatin cdna / mrna gene sequence . the corresponding sequences complementary to the genbank human myostatin sequences are as follows with differences between the genbank mouse and human underlined : the skipping efficiency of these aos can be tested by transfection ( leashed or unleashed : concentration between 50 and 500 nm ) into cultured human myoblast cells ( eg using a transfection reagent such as lipofectamine2000 ™), and evaluation of skipped and unskipped mrnas by electrophoretic densitometric analysis of rtpcr reaction products . 1 . mcpherron , a c , lawler , a m and lee , s j ( 1997 ). regulation of skeletal muscle mass in mice by a new tgf - β superfamily member . nature 387 : 83 - 90 . 2 . schuelke , m , wagner , k r , stolz , l e , hübner , c , riebel , t , kömen , w et al . 2004 ). myostatin mutation associated with gross muscle hypertrophy in a child . n engl j med 350 : 2682 - 2688 . 3 . bogdanovich , s , perkins , k j , krag , t o , whittemore , l a and khurana , t s ( 2005 ). myostatin propeptide - mediated amelioration of dystrophic pathophysiology . faseb j 19 : 543 - 549 . 4 . foster , k , graham , i r , otto , a , foster , h , trollet , c , yaworsky , p j et al . 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