Patent Application: US-201214240832-A

Abstract:
the present invention discloses cell penetrating peptides and their conjugates with cargo molecules . the peptides are useful as drug delivery systems , particularly as delivery vehicles for nucleotide - based theraputics , such as polynucleotides , oligonucleotides and peptide nucleic acids . a cpps of the invention provides a balance between good cell entry efficency and low toxicity and comprises three contiguous domains : the central one being hydrophobic and the flanking ones consisting of arginine and aminohexanoic acid or beta - alanine residues . the hydrophobic domain contains a sequence selected from yqfli , yrfli , iqfli and irfli .

Description:
the details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention , by way of example . it will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details . the inventors have designed a further series of cell penetrating peptides designed for conjugation to pmo to investigate the role of the central hydrophobic core sequence in obtaining high exon skipping and dystrophin production in the heart muscles in a mdx mouse model of duchenne muscular dystrophy ( dmd ) whilst maintaining the high activity across all muscles types compared to already disclosed pip5e - pmo . in particular the inventors have sought to design a cpp that a ) exhibits enhanced delivery of pmo cargo in heart muscle , and b ) has low toxicity . in view of this , the inventors have designed a series of peptides that differ in two main aspects from the previously disclosed pip - 2 to pip - 5 series . 1 ) the sequence of the domain 2 hydrophobic core amino acids has been inverted , or scrambled . 2 ) the number of arginine residues has been systematically varied to determine effect on cargo delivery and cellular toxicity . we describe a series of peptides designed for conjugation to pmo to investigate the role of the central hydrophobic core in obtaining high exon skipping and dystrophin production in the heart muscles in an mdx mouse model of duchenne muscular dystrophy ( dmd ) whilst maintaining the high activity across all muscles types compared to the already disclosed pip5e - pmo . we have previously reported impressive heart activity including high splicing efficiency and dystrophin restoration following a single administration of an arginine - rich cell - penetrating peptide conjugated to a phosphorodiamidate morpholino oligonucleotide : pip5e - pmo . however , the mechanisms underlying this activity are poorly understood . here , we report the results of studies involving single dose administration ( 12 . 5 mg / kg ) of a series of derivatives of pip5e - pmo , consecutively assigned as pip6 - pmos , containing mutations to the hydrophobic core region of the pip5e peptide , where this central core region amino acid sequence is reversed , scrambled or partially deleted . these changes affect the levels of exon skipping and dystrophin restoration in multiple muscle groups , including the heart , following a single , low dose intravenous injection of the corresponding pip6 - pmo conjugates . the results show that a core length of 5 amino acids ( 5 - aa ) appears to be essential for heart dystrophin production , since reductions in core length reduced cardiac activity . unexpectedly , an arginine residue was ( partially ) tolerated in one position of the hydrophobic core , but two arginine residues were not tolerated , nor an arginine in a different position . surprisingly , skeletal dystrophin production was also reduced in these two latter cases . our data indicate that the hydrophobic core of the pip sequences is critical for pmo delivery to the heart and that specific modifications to this region can enhance activity further . the results have implications for therapeutic pmo development for dmd . our previous lead pip series cpp , pip5e [ yin , h ., et al ., pip 5 transduction peptides direct high efficiency oligonucleotide - mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice . mol ther , 2011 . 19 ( 7 ): p . 1295 - 303 ], contains two arginine - rich flanking regions and a central hydrophobic core . to further probe the composition requirements of the hydrophobic core for maintenance of good heart dystrophin production , we synthesized a range of pip5e derivative peptides ( pip6 a - f ) where mutations were made only to the hydrophobic core region , for example scrambled and partially deleted core region peptides . all peptides contained the same number of arginine residues ( 10 ) in the flanking sequences as in pip5e , with the exception of pip6e . these peptides were conjugated to a 25 - mer pmo complementary to dystrophin exon 23 [ yin , h ., et al ., cell - penetrating peptide - conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function . hum mol genet , 2008 . 17 ( 24 ): p . 3909 - 18 ; yin , h ., et al ., a fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin - deficient mdx mice . hum mol genet , 2009 . 18 ( 22 ): p . 4405 - 14 . ], previously validated for exon skipping in mdx mice . in contrast to the method of conjugation to the 5 ′ end of pmo that we utilised previously [ yin et al supra ], pip6 - pmo conjugates were prepared by conjugation of the 3 ′ end of the pmo to the c - terminal carboxylic acid moiety of the pip peptide . we reported that there was no significant difference between the in vivo dystrophin production or exon skipping activity for pip5e - pmo conjugated to the 3 ′ end of the pmo or to the 5 ′ end and therefore chose to utilise 3 ′ end conjugation for these experiments [ saleh a f , a . a . a ., yin h , betts c , hammond s , wood m j a and gait m j , enhancement of exon skipping and dystrophin production by 3 ′- peptide conjugates of morpholino ( pmo ) oligonucleotides in a mdx mouse model of duchenne muscular dystrophy in collection symposium series , chemistry of nucleic acid components . 2011 , institute of organic chemistry and biochemistry , academy of sciences of the czech republic : prague . p . 292 - 296 ]. pmo 25 - mer m23d (+ 7 - 18 ) ( 5 ′- ggccaaacctcggcttacctgaaat - 3 ′ [ seq id no : 310 ]) ( pmodmd ) is commonly used as an oligonucleotide analogue suitable for exon skipping in mdx mice 37 , 42 , 43 , 44 . in many of these examples , b peptide ( sequence rxrrbrrxrrbrxb [ seq id no : 311 ]) was conjugated to pmodmd and shown to significantly enhance exon skipping and dystrophin production by intramuscular or intravenous delivery in mdx mice compared to naked pmodmd . the b - peptide ( which differs from ( rxr ) 4 xb by only two replacements of x by b units ) is a leading candidate peptide for clinical trial development in conjugation with a pmo for dmd treatment 43 . the peptides were synthesized by standard fmoc - based solid phase synthesis on a liberty microwave peptide synthesizer ( cem ). peptides were cleaved from the resin by treatment with trifluoroacetic acid ( tfa , 94 %) in the presence of triisopropylsilane ( 1 %), 1 , 2 - ethanedithiol ( 2 . 5 %) and water ( 2 . 5 %), purified by reversed phase hplc and analysed by maldi - tof mass spectrometry on an applied biosystems voyager de - pro using a matrix of β - cyano - 4 - hydroxycinnamic acid ( 10 mg / ml ) dissolved in acetonitrile / 3 % aqueous tfa ( 1 : 1 , v / v ). the amino acid sequences of peptides pip - 6a to pip - 6i , pip - 7a to pip - 7c2 and pip8a to pip - 8c2 are shown in fig1 . pip - 6a to pip - 6i , pip - 7a to pip - 7c2 and pip8a to pip - 8c2 and control b peptide were synthesized as conjugates of pmodmd . peptides were conjugated via an amide coupling reaction of a c - terminal carboxylic acid to the 3 ′- end of a 25 - mer pmo ( see yin et al . molecular therapy vol . 19 , no . 7 1295 - 1303 , july 2011 ). briefly , 3 ′- amide linkage was carried out as follows : pmo ( 100 nmole ) in dmso was coupled with a 2 - fold excess of peptide using tbtu : hoat : diea ( 2 . 5 : 1 . 8 : 1 . 7 molar excess over peptide ) in nmp at 37 ° c . for 2h . the conjugate was purified by cation exchange hplc ( source 15s , ge healthcare ) and desalted on a hlb column ( waters ). cell culture exon skipping assays in differentiated h2k mdx muscle myotubes the exon skipping potential of pip6 - pmo conjugates was evaluated in differentiated mouse h2k mdx myotubes in the absence of any transfection agent ( fig1 ) at concentrations ranging from 0 . 125 μm to 1 μm . this showed that exon skipping activity in cultured muscle cells was very similar for all these constructs , including pip5e - pmo . these results differ from the previous pip5 series [ yin et al supra ], where the flanking arginine - rich sequences mostly contained a fixed number of arginine residues ( 10 ) but where spacings were varied through alternative placement of aminohexanoyl ( aminohexanoic acid ) and β - alanine units . this resulted in small variations in exon skipping activity that correlated well with in vivo activity . in the case of pip6 sequences , the flanking arginine - rich sequences are identical ( with the exception of pip6e , which is identical except for one arginine immediately preceding the core which is displaced into the second position of the core ). the results demonstrate that cellular exon skipping activity does not depend on the sequence or length of the hydrophobic core . note that we have previously shown that major changes in in vitro exon skipping activities are correlated instead with the total numbers of arginine residues [ saleh , a . f ., et al ., synthesis and splice - redirecting activity of branched , arginine - rich peptide dendrimer conjugates of peptide nucleic acid oligonucleotides . bioconjug chem , 2010 . 21 ( 10 ): p . 1902 - 11 .]. h2k mdx myotubes are derived from h2k mdx myoblasts obtained from the edl muscle of a male h2k mdx f2 mouse . the cells are dystrophin deficient and conditionally immortal due to thermolabile simian virus 40 large tumor antigen ( tsa58 ) expression . the myoblasts proliferate at 33 ° c . ( 10 % co 2 ) in rich culture and differentiate into myotubes at 37 ° c . in media with horse serum . h2k mdx myotubes were prepared and incubated with peptide - pna and peptide - pmo conjugates in the absence of any transfection agent by the methods described previously ( wang , q , yin , h , et al . ( 2010 ) in vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for duchenne muscular dystrophy ) but with minor variations . myotubes were obtained from confluent h2k mdx cells seeded in gelatine coated 24 - well plates following 2 days of serum deprivation ( dmem with 5 % horse serum ). the conjugates were incubated with myotubes for 4 h in 0 . 5 ml optimem and then replaced by 1 ml of dmem / 5 % horse serum media for further incubation . after 20 h myotubes were washed twice with pbs and total rna was extracted with 0 . 5 ml of tri reagent ( sigma , uk ). rna preparations were treated with rnase free dnase ( 2u ) and proteinase k ( 20 mg ) prior to rt - pcr analysis . rt - pcr was carried out in 25 μl with 1 μg rna template using superscript iii one - step rtpcr system with platinum taq dna polymerase ( invitrogen ) primed by forward primer 5 ′ cag aat tct gcc aat tgc tgag3 ′ [ seq id no : 312 ] and reverse primer 5 ′ ttc ttc agc ttg tgt cat cc3 ′ [ seq id no : 313 ]. the initial cdna synthesis was performed at 55 ° c . for 30 min followed by 30 cycles of 95 ° c . for 30 sec , 55 ° c . for 1 min and 68 ° c . for 80 sec . rt pcr product ( 1 ml ) was then used as the template for secondary pcr performed in 25 μl with 0 . 5 u super taq polymerase ( ht biotechnologies ) and primed by forward primer 5 ′ ccc agt cta cca ccc tat cag agc3 ′ [ seq id no : 314 ] and reverse primer 5 ′ cct gcc ttt aag gct tcc tt3 ′ [ seq id no : 315 ]. the cycling conditions were 95 ° c . for 1 min , 57 ° c . for 1 min and 72 ° c . for 80 sec for 25 cycles . products were examined by 2 % agarose gel and after scanning using gene tools analysis software ( syngene ) the relative amount of exon 23 skipping was expressed as a percentage at a given concentration of conjugates averaged over duplicates of three experiments . the results ( fig1 ) showed that all of the pip - 6a - pmo , pip - 6b - pmo , pip - 6c - pmo , pip - 6d - pmo , pip - 6e - pmo and pip - 6f - pmo conjugates gave high exon skipping activity in mdx muscle cells . pip - 6g - pmo , pip - 6h - pmo , pip - 6i - pmo , pip - 7a - pmo , pip - 7b - pmo , pip - 7b2 - pmo , pip - 7c2 , pip - 8a - pmo , pip - 8b , and pip - 8c conjugates also gave high exon skipping activity in mdx muscle cells and pip 7c and pip7d gave moderate exon skipping ( fig2 - 5 ). given the potency of pip5e - pmo in heart tissue , the aim of altering the sequence of the hydrophobic core ( whilst maintaining the 5 - aa length ) was to identify peptides that might be more efficient at lower doses . these modifications included inversion of the hydrophobic region ( pip6a ), substitution of tyrosine by isoleucine ( pip6b ), substitution of glutamine in the pip6a sequence by displacement of the arginine immediately flanking the core in the first arginine - rich flanking region ( pip6e ), and a scrambled hydrophobic core sequence ( pip6f ). all pip6 peptide - pmo conjugates were administered to mdx mice as single 12 . 5 mg / kg intravenous injections via the tail vein and tissues were harvested 2 weeks later and assessed for activity at both rna and protein levels . immunohistochemical staining of dystrophin expression for all 5 - aa core pip6 - pmos revealed high levels of dystrophin production in skeletal muscles including the ta , diaphragm , and the heart . immunohistochemical staining quantification ( fig3 a , b ) was performed as previously described [ malerba , a ., et al ., chronic systemic therapy with low - dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice . mol ther , 2011 . 19 ( 2 ): p . 345 - 54 ; arechavala - gomeza , v ., et al ., immunohistological intensity measurements as a tool to assess sarcolemma - associated protein expression . neuropathol appl neurobiol , 2010 . 36 ( 4 ): p . 265 - 74 .] and was achieved by taking 4 representative frames of the dystrophin staining and correlating this with laminin staining for each section ( n = 3 ) of the quadriceps , diaphragm and heart for each peptide - pmo treatment . untreated mdx and treated mdx mice were normalised to c57bl10 mice . this method allows comparison of the staining intensity of dystrophin at the sarcolemma relative to laminin for each treatment group . intensity ratios are normalised to c57bl10 samples and each region of interest at the sarcolemma ( 120 regions for each treatment group ) is plotted on a scatter graph . the relative intensity values obtained for all four of the 5 - aa core pip6 - pmo conjugates were significantly different to those of untreated mdx mice for the quadriceps and diaphragm ( fig3 b and fig3 ). there were very similar dystrophin restoration levels in the quadriceps ( percent recovery score -% rs - range between 21 . 10 - 33 . 44 %; fig3 b ) and in the diaphragm for all treatments , with the exception of pip6b which had a higher recovery score in the diaphragm (% rs range between 38 . 87 - 48 . 43 %, pip6b 56 . 72 %; fig3 b ). all 5 - aa core pip6 - pmo - treated mice exhibited high dystrophin intensity values in the heart with the exception of pip6e ( other pip6 - pmos were statistically significant compared to mdx = p & lt ; 0 . 0001 ; fig3 ). pip6a - and pip6b - pmo conjugates displayed the highest recovery scores , as observed in fig3 b , (% rs 37 . 66 % and 34 . 22 % respectively ) closely followed by pip6f - pmo (% rs 26 . 24 %) and then pip5e - pmo (% rs 17 . 32 %). when directly compared to pip5e - pmo treatment , only pip6a - pmo was significantly better in the heart ( fig3 ). these 5 - aa core pip6 - pmos were also shown to restore other dystrophin complex proteins , namely nnos , α - sarcoglycan and β - sarcoglycan as illustrated by immunohistochemical staining in the ta muscle . the pcr and western blot analyses exhibited similar results to the immunostaining . the rtpcr representative images ( fig3 d ) illustrate high exon skipping efficiency in all tissues analysed . this is better shown by real time pcr ( qrt - pcr ) results for the quadriceps , diaphragm and heart ( fig3 c ). the delta 23 transcript is normalised against ‘ total dystrophin ’ for each muscle group ( n = 3 ). quantification of this data revealed similar levels of δ23 skipping in the quadriceps of all 5 - aa pip6 - pmo treated mice . the data trends suggest that pip6f - and pip5e - pmo show the highest exon skipping in the diaphragm , and pip6f - pmo the highest in heart ( for splicing mean values see fig3 a ). western blots ( fig3 e ) were performed on the tissues of each mouse and were quantified against a 50 % and 10 % c57bl10 control . these results were averaged and are presented in fig3 b . pip6a -, pip6b -, pip6e - and pip6f - pmo conjugates exhibited the highest dystrophin protein restoration in the ta and quadriceps muscles . the levels of dystrophin restoration in the diaphragm were uniform across all of these treatments , whereas in the case of the heart , pip6b - and pip6f - pmo conjugates showed the highest dystrophin restoration . protein restoration as measured by immunohistochemical staining is consistently higher than protein restoration calculated by western blot analysis . these differences may be attributed to the differing ‘ housing proteins ’ used i . e . dystrophin restoration quantified by immunohistochemical staining is normalised against laminin , whereas western blot analysis uses alpha - actinin for normalisation . quantification of western blots has only recently been reported for dystrophin and currently uses chemiluminescence methods . it may therefore be judicious to give greater weight to the trends in dystrophin protein levels revealed by western blot rather than to the absolute values . therefore , considering the results overall , mdx mice treated with each of the four 5 - aa core pip6 - pmos ( pip6a -, pip6b -, pip6e - and pip6f - pmo ) appear to demonstrate improved dystrophin production and exon skipping in ta , quadriceps and heart muscles compared with the previous lead candidate , pip5e - pmo . in addition , these 5 - aa core pip6 - pmos do not exhibit evidence of toxicity , as assessed by plasma levels of relevant toxicity biomarkers , alanine aminotransferase ( alt ), aspartate aminotransferase ( ast ) and creatine kinase ( see fig4 a ). blood urea nitrogen and creatinine levels were similar to untreated mdx levels ( see fig4 b ). all pip6 - pmo treatment groups exhibit similar biomarker levels to untreated c57bl10 controls . as described above , pip5e - pmo was compared to pip6 - pmo conjugates for exon skipping and dystrophin production in adult mdx mice ( 4 . 5 months old ) treated with a single intravenous injection of 12 . 5 mg / kg and body wide tissues were harvested 2 weeks later . immunohistochemical staining ( data not shown ) was used to visualise the production of dystrophin protein ( and its correct re - location at the sarcolemma ) and rt - pcr was used to detect exon - skipped products in treated mdx muscle groups . qpcr allows for the quantification of the spliced product and western blots were used to quantify the amount of dystrophin protein produced in muscles from treated mdx mice as compared with c57bl10 and untreated mdx controls . the results ( fig6 - 17 ) showed that pip - 6a ( containing an inverted core ( domain 2 ) sequence from pip - 5e ), pip - 6b ( y changed to i in the core sequence ), pip - 6e ( r moved into the core sequence ) and pip - 6f ( containing a scrambled core sequence ) all maintained the exon skipping and dystrophin production in heart muscle similarly to pip - 5e , whereas peptides containing a truncated core sequence ( pip - 6c and pip - 6d ) lost activity in heart muscle . activity in all other muscle types was broadly similar for all six pip - 6 ( a , b , c , d , e , f ) pmo constructs . these results indicate that for efficient delivery of pmodmd to heart muscle the peptide sequence is not important , which suggests there is not a specific cell entry signal ( e . g . scrambled core : pip - 6f exhibits good delivery of pmodmd to heart muscle ). thus , whilst retention of a 5 amino acid hydrophobic core sequence is important , the n - to c - order ( primary sequence ) of that hydrophobic core sequence is not important as demonstrated by inversion and scrambling of the core sequence without loss of activity in heart muscle compared to pip - 5e . however , for delivery to other muscle types , e . g . skeletal and smooth muscle , the requirement to have a 5 amino acid hydrophobic core sequence is less important , as demonstrated by good activity in non - cardiac muscle by pip - 6c and pip - 6d . huh - 7 cells were grown at 37 ° c . under 5 % co 2 / 95 % air atmosphere in dmem supplemented with 10 % fetal bovine serum and penicillin / streptomycin antibiotics . cells were treated with trypsin and plated at 1 . 5 × 10 4 cells per well in 96 well plates . after overnight incubation cells were washed with pbs and pmo - peptide conjugates in 50 μl optimem were added to the wells in triplicate . after 4 hours incubation , conjugates were removed by replacement of media with 100 μl dmem / 10 % fbs for a further 20 h incubation . 20 μl of mts cell viability assay ( promega ) solution was added to the wells containing 100 μl dmem and plates were incubated for 1 - 2 hours before the uv measurements at 490 nm were taken . the cell viability percentage was determined by normalizing the average absorbance of triplicate samples to the mean of untreated samples . results presented ( fig1 - 22 ) are the average of two independent experiments , and are consistent with increased cell viability ( i . e . lower cellular toxicity ) of peptides containing fewer arginine residues . for example , peptides having a total of 7 arginine residues in domains 1 - 3 combined exhibited higher cell viability ( lower cellular toxicity ) than peptides having 8 or 9 arginine residues . serum stability was measured by adding an aliquot of pip - pmo ( 10 nmoles ) to 100 % mouse serum ( 100 μl ) and incubating at 37 ° c . for 120 or 240 min . each reaction was diluted with im guanidinium - hcl solution ( 300 μl ) and ice - cold acetonitrile ( 600 μl ). samples were mixed and kept at − 20 ° c ., and the precipitated serum proteins separated by centrifugation ( 13000 rpm , 5 min ). after centrifugation , the supernatant was collected , lyophilized and the residue dissolved in water for analysis for peptide degradation by maldi - tof mass spectrometry and by reversed phase hplc . hplc conditions were : column , c18 reversed - phase ( 250 × 4 . 6 mm ); solvent a , 0 . 1 % tfa , solvent b , 90 % acetonitrile , 10 % solvent a ; gradient , 10 %- 50 % solvent b in 25 minutes . the results showed that for pip5e - pmo , pip6a - pmo , pip6e - pmo and pip6f - pmo at 120 minutes , the main component remaining could be identified as the unchanged pip - pmo conjugate and that the levels of pmo conjugated to fragments of peptide were very similar to that for ( rxrrbr ) 2 xb - pmo . partial deletions of the hydrophobic core of pip6 - pmo conjugates abolish heart dystrophin production in addition to the need to identify pip - pmos with high efficiency and cardiac delivery , it was a further aim to better define those elements of the hydrophobic core of pip peptides that are important for heart delivery . to this end , pip peptides containing partial deletions of the hydrophobic core by 1 amino acid ( removal of tyrosine ; pip6c ) and by 2 amino acids ( removal of isoleucine and tyrosine ; pip6d ) were synthesised as pmo conjugates . following treatment of mdx mice , immunohistochemical staining was performed and this revealed some dystrophin expression in skeletal muscles such as the ta and diaphragm for both these deletion pip6 - pmos . quantification of the immunohistochemical staining revealed the lowest dystrophin restoration in the quadriceps with pip6d - treatment , closely followed by pip6c - pmo when compared to the other pip6 - pmos ( fig3 a , b and fig3 ). similarly , pip6d - pmo displayed the lowest dystrophin restoration in the diaphragm . the recovery scores for pip6c - and pip6d - pmo conjugates in the heart were very low , indicating their poor efficiency ( pip6c % rs − 1 . 06 % and pip6d 2 . 50 %; fig3 b ). these results are corroborated by the pcr and western blot analyses . the rt - pcr representative images ( fig3 d ) and the qrt - pcr exon skipping results ( fig3 c ) both indicate reduced exon skipping in mdx mice treated with pip6c - and pip6d - pmo conjugates in quadriceps and diaphragm and negligible exon skipping in the heart . western blot analysis revealed inefficient dystrophin protein production in the ta and quadriceps muscles and negligible dystrophin restoration in the heart ( fig3 e and fig3 b ). these results show that the length of the hydrophobic core is crucial not only for good heart dystrophin production but also for activity in some other muscle groups . therefore the arginine content of the cpp alone is not the sole predictor of dystrophin production and exon skipping efficiency for this class of peptides . altering the position of arginine in the hydrophobic core or adding a second arginine is detrimental to dystrophin production the repositioning of an arginine from a flanking region into the core was unexpectedly tolerated ( pip6e - pmo ). two further pip6 - pmo conjugates were thus synthesized as derivatives of pip6e - pmo . pip6g - pmo contained a second arginine residue , which was moved from the second flanking region into the central hydrophobic core , and pip6h - pmo contains an inversion of the pip6e hydrophobic region , such that the single arginine location is altered within the core . surprisingly , these changes to the hydrophobic core resulted in further reductions in dystrophin expression not only in heart but also in all other tissues , as observed in the immunohistochemical staining and in the quantifications thereof ( fig3 a , b ). the immunohistochemical staining representative images revealed very few dystrophin positive fibres in all tissues with the exception of the ta and quadriceps . with reference to the quantifications , pip6g - pmo was not significantly different to untreated mdx in the quadriceps or diaphragm ( fig3 a ). both pip6e - pmo derivatives were not significantly different to untreated mdx in heart muscles , illustrating the general inefficiency of these two peptides . similarly , these pip6e - pmo derivatives showed reduced efficiency in exon skipping , as illustrated in representative rt - pcr images ( fig3 d ) and in qrt - pcr analyses ( fig3 c and fig3 c ) in all tissues . western blots revealed negligible dystrophin protein restoration ( fig3 e and fig3 d ) in all tissues with the exception of the ta muscle . these data show that an increase in the number of arginines or alteration in the location of the single arginine in the hydrophobic region of pip6e are detrimental to both heart as well as skeletal muscle dystrophin production . peptides were synthesized by standard fmoc chemistry and purified by hplc . the pmo sequence ( 5 ′- ggccaaacctcggcttacctgaaat - 3 ′ [ seq id no : 310 ]) was purchased from gene tools llc . peptides were conjugated to pmo through an amide linkage at the 3 ′ end of the pmo , followed by purification by hplc and analysed by maldi - tof ms as previously described in preliminary communication [ saleh a f , a . a . a ., yin h , betts c , hammond s , wood m j a and gait m j , enhancement of exon skipping and dystrophin production by 3 ′- peptide conjugates of morpholino ( pmo ) oligonucleotides in a mdx mouse model of duchenne muscular dystrophy in collection symposium series , chemistry of nucleic acid components . 2011 , institute of organic chemistry and biochemistry , academy of sciences of the czech republic : prague . p . 292 - 296 ]. peptide - pmo conjugates were dissolved in sterile water and filtered through a 0 . 22 μm cellulose acetate membrane before use . conjugates of pmo of pip6a , pip6b , pip6e and pip6f were found to be predominantly stable and of similar stability to pip5e - pmo in 100 % serum for 2 h at 37 ° c ., as seen by hplc and maldi - tof mass spectral analysis . the conjugates all showed similar degradation patterns , and intact conjugates were still observed up to 4 h , ( data not shown ). h2k mdx myotubes were prepared and incubated with peptide - pmo conjugates in the absence of any transfection agent at concentrations of 0 . 125 , 0 . 25 , 0 . 5 and 1 . 0 μm by the method described previously [ yin , h ., et al ., pip 5 transduction peptides direct high efficiency oligonucleotide - mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice . mol ther , 2011 . 19 ( 7 ): p . 1295 - 303 .]. the products of nested rt - pcr from total isolated rna were examined by electrophoresis on a 2 % agarose gel . quantification of δ23 transcript levels was calculated using densitometry . the mts cell viability test ( promega ) showed 100 % survival at the highest concentrations of peptide - pmo conjugates used in the study ( data not shown ). four and a half month old to 5½ month old mdx mice were used in these experiments ( n = 3 ). the experiments were carried out in the biomedical sciences unit , university of oxford according to procedures authorised by the uk home office . pip6 - pmo conjugates were prepared in 0 . 9 % saline solution at a final dose of 12 . 5 mg / kg . the 160 μl total volume was administered via the tail vein of anaesthetised mice . two weeks later mice were sacrificed by co 2 inhalation , and muscles and other tissues harvested and snap - frozen in cooled isopentane before storage at − 80 ° c . transverse sections of tissue samples were cut ( 8 μm thick ) for the examination of dystrophin expression . for dystrophin visualisation and quantification , sections were co - stained with rabbit - anti - dystrophin ( abcam ) and rat anti - laminin ( sigma ), and detected by goat - anti - rabbit igg alexa 594 and goat - anti - rat igg 488 secondary antibodies respectively ( invitrogen ). images were captured using a leica dm irb microscope and axiovision software ( carl zeiss ). quantitative immunohistochemistry was performed as previously described [ malerba , a ., et al ., chronic systemic therapy with low - dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice . mol ther , 2011 . 19 ( 2 ): p . 345 - 54 ; arechavala - gomeza , v ., et al ., immunohistological intensity measurements as a tool to assess sarcolemma - associated protein expression . neuropathol appl neurobiol , 2010 . 36 ( 4 ): p . 265 - 74 .]. a representative image for each treatment was taken . for quantification , 4 representative frames of the dystrophin and correlating laminin were taken for each section ( n = 3 ) of the quadriceps , diaphragm and heart for each treatment . using imagepro software , 10 regions of interest were randomly placed on the laminin image which was overlaid on the corresponding dystrophin image . the minimum and maximum fluorescence intensity for 120 regions were recorded for each treatment . the intensity difference was calculated for each region to correct for background fluorescence and untreated mdx and treated mdx were normalised to c57bl10 . these values were plotted on a scatter graph . the ‘ relative intensity means ’ were calculated using a multi - level statistics model . using these values , the percentage recovery score was calculated by implementing the following equation , as described on the treatnmd website ( http :// www . treatnmd . eu / downloads / file / sops / dmd / mdx / dmd_m . 1 . 1 — 001 . pdf ): ( dystrophin recovery of treated mdx mice - dystrophin recovery of untreated mdx mice )/( dystrophin recovery of c57bl10 mice - dystrophin recovery of untreated mdx mice ). staining of dystrophin associated proteins was performed as previously described [ yin , h ., et al ., pip 5 transduction peptides direct high efficiency oligonucleotide - mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice . mol ther , 2011 . 19 ( 7 ): p . 1295 - 303 ] using a mom blocking kit ( vector labs ) and α - sarcoglycan and α - dystroglycan ( novocastra ) antibodies ( 1 : 100 dilution ). nnos staining was performed using a goat anti - rabbit antibody ( abcam ). total rna was extracted from control and treated mouse tissues using trizol reagent ( invitrogen ) following manufacturer &# 39 ; s instructions . four hundred nanograms of rna template was used in a 50 μl reverse transcription reaction using one step rt - pcr kit ( qiagen ) and gene specific primers ( ex 20 - 26 , fwd : 5 ′- cag aat tct gcc aat tgc tga g - 3 ′ [ seq id no : 312 ], rev : 5 ′- ttc ttc agc ttg tgt cat cc - 3 ′ [ seq id no : 313 ]). cycle conditions : 50 ° c . for 30 minutes , followed by 30 cycles of 30 sec at 94 ° c ., 1 min at 58 ° c ., and 2 min at 72 ° c . two microlitres of cdna was further amplified in a 50 μl nested pcr ( qiagen pcr kit ) using the following cycle conditions : 94 ° c . for 30 seconds , 58 ° c . for 1 minute , and 72 ° c . for 1 min for 24 cycles ( ex 20 - 26 : fwd : ccc agt cta cca ccc tat cag agc [ seq id no : 314 ], rev : cct gcc ttt aag gct tcc tt [ seq id no : 315 ]). pcr products were examined by electrophoresis on a 2 % agarose gel . two micrograms of rna was reverse transcribed using a high capacity cdna synthesis kit ( applied biosystems ). exon skipping qpcr was performed using syber green kits ( applied biosystems ), primer sets ( idt ) and the stepone plus real - time pcr system ( applied biosystems ). primer sets used were as follows : total dystrophin transcripts , ex19 - 20 : fwd : gccatagcacgagaaaaagc [ seq id no : 812 ], rev : gcattaacaccctcatttgc [ seq id no : 813 ]; delta23 dmd transcript , fwd : gcg cta tca gga gac aat gag [ seq id no : 814 ], rev : gtt ttt atg tga ttc tgt aat ttc cc [ seq id no : 815 ]. plasmids ( total dystrophin and delta 23 skipped ) were used for the standard curve . control and treated muscle samples were homogenised in lysis buffer comprising 75 mm tris - hcl ( ph 6 . 5 ) and 10 % sodium dodecyl sulphate complemented with 5 % 2 - mercaptoethanol . samples were heated at 100 ° c . for 3 minutes before centrifugation and removal of supernatant . protein levels were measured by bradford assay ( sigma ) and quantified using bsa standards . ten to 15 μg of protein of untreated and treated mdx sample , and 50 % and 10 % of these concentrations of c57bl10 protein ( positive control ) were loaded onto 3 - 8 % tris - acetate gels . proteins were blotted onto pvdf membrane and probed for dystrophin using dys1 ( novocastra ) and loading control , α - actinin ( sigma ), antibodies . primary antibody was detected by binding of horseradish peroxidise - conjugated anti - mouse igg with lumigen . western blots were imaged ( licor biosciences ) and analysed using the odyssey imaging system . plasma samples were taken from the jugular vein of mdx mice immediately following sacrifice by co 2 inhalation . analysis of toxicity biomarkers was performed by a clinical pathology lab , mary lyon centre , mrc , harwell , uk . all data reported mean values ± sem . a multi - level , repeated measures model was implemented for this study . the multi - level statistical approach builds upon traditional statistical methods and is being increasingly implemented in the social , medical and biological sciences [ butterfield , a ., et al ., pyevolve : a toolkit for statistical modelling of molecular evolution . bmc bioinformatics , 2004 . 5 : p . 1 ; brooks , g ., et al ., referral source and outcomes of physical therapy care in patients with low back pain . j orthop sports phys ther , 2012 ; bernier , j ., y . feng , and k . asakawa , strategies for handling normality assumptions in multilevel modeling : a case study estimating trajectories of health utilities index mark 3 scores . health rep , 2011 . 22 ( 4 ): p . 45 - 51 ; winter , e . m ., r . g . eston , and k . l . lamb , statistical analyses in the physiology of exercise and kinanthropometry . j sports sci , 2001 . 19 ( 10 ): p . 761 - 75 .]. the model used for this study takes into account the multiple ‘ relative intensity units ’ ( level 1 ) for each mouse ( level 2 ) for each treatment ( level 3 ) as performed in the immunohistochemical staining quantification . in this example mdx untreated mice and pip5e - pmo treated mice were applied as the constant / fixed parameter , to which the other treatments and wild - type control were compared . this was following a box - cox power transformation which was performed to ensure a normal distribution . statistical analysis was performed using mlwin version 2 . 25 . the pmo sequence ( 5 ′- ggccaaacctcggcttacctgaaat - 3 ′ [ seq id no : 310 ]) was purchased from gene tools llc . peptides were synthesized by standard 9 - fluorenylmethoxy carbonyl ( fmoc ) chemistry , using a liberty peptide synthesizer ( cem ) on 100 μmole scale , purified to & gt ; 90 % purity by standard reversed phase hplc , and characterized by maldi - tof ms . peptide - pmo conjugates were prepared on 200 nmole pmo scale via an amide linkage by attaching the carboxyl group at the c - terminus of peptide to the secondary amine at the 3 ′- end of the pmo ( fig1 c ). the c - terminal carboxylic acid of peptide ( 2 . 5 - fold molar excess over pmo ) was activated using 2 -( 1h - benzotriazole - 1yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluorophosphate ( hbtu ) and 1 - hydroxybenzotriazole ( hobt ) in 1 - methyl - 2 - pyrrolidinone ( nmp ) and diisopropylethylamine ( diea ) using a hbtu : hobt : diea ( 2 . 3 : 2 . 0 : 2 . 3 ) molar excess over peptide . the mixture was added to a solution of pmo ( 10 mm ) dissolved in dmso and incubated at 37 ° c . for 2 h . the mixture was then diluted with a 4 - fold excess of water and purified on a cation exchange chromatography column ( resource s 6 - ml column , ge healthcare ) using sodium phosphate buffer ( buffer a : 25 mm na 2 hpo 4 , 25 % acetonitrile , ph 7 . 0 ), buffer b : 1 m nacl , 25 mm na 2 hpo 4 , 25 % acetonitrile , ph 7 . 0 ), a flow rate of 4 ml / min and a gradient of 0 - 75 % buffer b in 25 min to remove unconjugated pmo and excess peptide . the conjugates were then loaded onto an oasis hlb column ( 4 . 6 mm × 20 mm , waters , milford , mass . ), washed with water to remove salts and eluted with 60 % ( v / v ) acetonitrile . the conjugate was lyophilized and analysed by maldi - tof ms and by hplc ( fig3 ). peptide - pmo conjugates were dissolved in sterile water and filtered through a 0 . 22 μm cellulose acetate membrane before use . overall yields were 20 - 25 % based on pmo . conjugations were improved on a 1000 nanomole scale of starting pmo , keeping the same ratio for hbtu : hobt : diea as described above . conjugation reactions were carried out using a microwave oven ( cem discover ) and the temperature of reaction was increased to 65 ° c ., leading to a decrease in reaction time to 15 min . the crucial purification steps were also improved in this process . the crude reaction mixture was purified by hplc using a larger ion - exchange column ( source 15s , ge healthcare , hr16 / 10 , 18 ml bed volume ) to remove the excess of peptide and unconjugated pmo , eluting with a solution of sodium chloride ( nacl , 1 . 0 m ) in sodium phosphate buffer ( ph 7 . 0 ) to obtain the chloride salt of the peptide - pmo . a desalting step was used to remove the excess nacl on a custom - made oasis hlb desalting column ( waters , 19 × 30 mm ). after washing the column for 10 - min with millipore grade water ( instead of hplc - grade water ), the ppmo was eluted with 60 % ( v / v ) acetonitrile in water . 500 nanomoles were used in a single injection and yields were higher than using multiple injections ( e . g . 2 × 250 nmol ). this resulted in an increase in yield of this one - pot conjugation reaction to approx 40 %. the most promising therapy to date for the severely debilitating neuromuscular disorder dmd is treatment with aos , which restores the reading frame of the dystrophin pre - mrna by exon skipping . two aos , a pmo [ cirak , s ., et al ., exon skipping and dystrophin restoration in patients with duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment : an openlabel , phase 2 , dose - escalation study . lancet , 2011 . 378 ( 9791 ): p . 595 - 605 ; miller , f ., c . f . moseley , and j . koreska , spinal fusion in duchenne muscular dystrophy . dev med child neurol , 1992 . 34 ( 9 ): p . 775 - 86 .] and a 2 ′ ome oligonucleotide [ van deutekom , j . c ., et al ., local dystrophin restoration with antisense oligonucleotide pro 051 . n engl j med , 2007 . 357 ( 26 ): p . 2677 - 86 ; goemans , n . m ., et al ., systemic administration of pro 051 in duchenne &# 39 ; s muscular dystrophy . n engl j med , 2011 . 364 ( 16 ): p . 1513 - 22 . ], are currently in clinical trials and the early promising results have increased hope for dmd patients . however , studies involving the administration of very high doses of naked pmo into mdx mice have shown only partial restoration of dystrophin in body - wide skeletal muscles and negligible correction in heart [ malerba , a ., et al ., chronic systemic therapy with low - dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice . mol ther , 2011 . 19 ( 2 ): p . 345 - 54 ; malerba , a ., et al ., dosing regimen has a significant impact on the efficiency of morpholino oligomer - induced exon skipping in mdx mice . hum gene ther , 2009 . 20 ( 9 ): p . 955 - 65 .]. the necessity to correct dystrophin in the heart is ever more apparent following studies whereby the correction of the skeletal phenotype resulted in an increase in the cardiac workload and thus further progression of the cardiomyopathy [ malerba , a ., l . boldrin , and g . dickson , long - term systemic administration of unconjugated morpholino oligomers for therapeutic expression of dystrophin by exon skipping in skeletal muscle : implications for cardiac muscle integrity . nucleic acid ther , 2011 . 21 ( 4 ): p . 293 - 8 ; townsend , d ., et al ., emergent dilated cardiomyopathy caused by targeted repair of dystrophic skeletal muscle . mol ther , 2008 . 16 ( 5 ): p . 832 - 5 .]. the discovery that cpp - conjugated pmos can achieve much more effective dystrophin correction in mdx mice than naked pmos has brought renewed promise for enhanced ao efficacy by improving cellular and in vivo delivery . we previously reported a promising peptide - pmo candidate , pip5e - pmo , capable of restoring dystrophin protein to high levels in all muscle types , including heart , following a single 25 mg / kg administration [ yin , h ., et al ., pip 5 transduction peptides direct high efficiency oligonucleotide - mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice . mol ther , 2011 . 19 ( 7 ): p . 1295 - 303 .]. in addition to arginine - rich sequences , pip peptides contain a 5 - aa hydrophobic section not present in the previous b - peptide lead [ perez , f ., et al ., antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide . j cell sci , 1992 . 102 ( pt 4 ): p . 717 - 22 . ], which seemed likely to be responsible for the improved heart activity . the pip6 series was developed as derivatives of pip5e - pmo in an attempt to cast light on aspects of the hydrophobic core required for heart dystrophin production and also to identify even more active pip - pmo conjugates . our study using a moderate , single dose administration regimen has produced some surprising results . a key finding is that maintenance of the five amino acid length of the hydrophobic core region is imperative for good heart dystrophin production . one might imagine that diminished efficiency of dystrophin restoration in the heart for pip6c - pmo and pip6d - pmo , with sequential amino acid deletions in the core , might be correlated with the resultant lower hydrophobicity and hence a reduced capacity to enter the cell [ gupta , a ., et al ., hydrophobicity drives the cellular uptake of short cationic peptide ligands . eur biophys j , 2011 . 40 ( 6 ): p . 727 - 36 .]. however , the in vitro results would suggest that all of these constructs are capable of entering the cells as they are all fully capable of exon skipping in muscle cells . thus , the length of the 5 - aa hydrophobic core must affect a different parameter essential for in vivo heart delivery . enhanced uptake into whole heart slices of fluorescently labelled pip5e - pmo , compared to b - pmo , suggested instead that crossing of another barrier ( for example the endothelial lining to the heart ) is improved [ yin , h ., et al ., cell - penetrating peptide - conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function . hum mol genet , 2008 . 17 ( 24 ): p . 3909 - 181 . further heart studies are continuing with a pip6 - pmo that may help to address this issue . more surprising perhaps is that for pip6c - pmo and pip6d - pmo there was also some loss of dystrophin production in other muscle types . this suggests that the hydrophobic / cationic balance and / or the precise spacings of hydrophobic and cationic residues in the cpp impose more subtle effects on in vivo delivery parameters . another clear conclusion arising from the pip6 - pmo analogues is that a specific order of hydrophobic residues within the hydrophobic core is less important at maintaining the heart dystrophin production , since an inverted sequence ( pip6a ), a single substitution of an equally hydrophobic residue ( pip6b ), and a scrambled sequence ( pip6f ) were at least as active as pip5e - pmo , and more efficient in heart and some muscle groups ( fig3 ). these results provide evidence that the hydrophobic core of pip peptides is unlikely to contain a particular amino acid sequence that recognises a specific receptor in a membrane barrier required to penetrate heart tissue , but instead the core acts as a hydrophobic spacer of some kind . most surprising however is that pip6e - pmo did induce some dystrophin splicing and protein restoration in heart muscle as indicated by the western and qrt - pcr results ( note : not significantly different in immunohistochemical staining quantification ). in the pip - 6e peptide , one arginine residue is moved into the hydrophobic core , which also results in alignment of a hydrophobic x residue adjacent to the core ( x - yrfli [ seq id no : 816 ]). one might have expected heart dystrophin production to have been completely lost in this conjugate , since a cationic amino acid ( arginine ) is now included in the core . by contrast , such heart activity was lost for pip6h - pmo ( x - ilfry core [ seq id no : 817 ]) and the double arginine core conjugate pip6g - pmo ( xyrfrli - x core [ seq id no : 818 ]). unexpectedly , dystrophin production was also lost in quadriceps and diaphragm for both pip6g - and pip6h - pmo . the unanticipated inconsistencies within the activity results for pip6e , pip6g and pip6h , and the losses of activities for pip6c - and pip6d - pmo , are perhaps best explained by the realisation that precise spacings of the arginine residue within the pip peptides with respect to both the outer hydrophobic amino acid spacers ( x and b ) and the inner hydrophobic core residues may drastically alter the pharmacological properties of each conjugate . this might occur not only through alteration in cationic / hydrophobic balance but alternatively due to secondary or tertiary structure changes of the pip - pmos , which could in turn affect serum protein binding or another parameter that alters the circulatory half - life , or which could affect the ability to traverse barriers required to penetrate muscle tissues . several conjugates ( pip6a , pip6b and pip6f - pmos ) have shown promising dystrophin production activities even beyond that of the previous candidate , pip5e - pmo . interestingly , analysis of serum samples from one of the 5 - aa pip6 - pmo treatments , pip6e - pmo , showed partial normalisation of 3 mirnas ( mir - 1 , mir - 133a and mir - 206 ) to near wild type levels following a single , 12 . 5 mg / kg administration [ thomas c . roberts , k . e . m . b ., graham mcclorey , samir e l andaloussi , caroline godfrey , corinne betts , thibault coursindel , michael j . gait , and c . i . e . s . a . m . j . a . wood , expression analysis in multiple muscle groups and serum reveals complexity in the microrna transcriptome of the mdx mouse with implications for therapy . nucleic acid ther , 2012 ]). this is greatly promising for the pip6 - pmos , as it would not be considered the optimal peptide yet still demonstrated the significant therapeutic effect of this group of peptides . these new leads provide a good basis for identification of a pip - pmo candidate suitable for detailed physiological studies of muscle and heart function , as well as thorough toxicity profiling including dose escalation studies , in anticipation that one such pip - pmo will proceed to clinical trial . the pmo sequence ( 5 ′- ggccaaacctcggcttacctgaaat - 3 ′) [ seq id no : 310 ] was purchased from gene tools , llc . peptides were synthesized in house by standard 9 - fluorenylmethoxy carbonyl ( fmoc ) chemistry , purified to & gt ; 90 % purity , and characterized by maldi - tof ms . peptide - pmo conjugates were prepared via an amide linkage by attaching the carboxyl group at the c - terminus of peptide to the secondary amine at the 3 ′- end of the pmo ( fig3 ). however , we made several modifications for conjugation and final purification of peptide - pmo including a ) large scale conjugation reaction ( up to 15 μmol of pmo ), b ) use of a larger scale ion exchange column for peptide - pmo purification from individual components ( table 1 ) and c ) the use of amicon ultra - 15 centrifugal filter device for desalting instead of the previously used reversed phase hplc column . as a result , yields were more than doubled and were routinely 40 - 55 % based on the pmo sequence . the c - terminal carboxylic acid of peptide ( 2 . 5 - fold molar excess over pmo ) was activated using 2 -( 1h - benzotriazole - 1yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluorophosphate ( hbtu ) and 1 - hydroxy - 7 - azabenzotriazole ( hoat ) in 1 - methyl - 2 - pyrrolidinone ( nmp ) and diisopropylethylamine ( diea ) using a hbtu : hobt : diea ( 2 . 3 : 2 . 0 : 2 . 3 ) molar excess over peptide ( table 2 ). the concentration of the peptide was 100 mm . after mixing , the mixture was added to a dmso solution of pmo ( 10 mm ) at 2 . 5 : 1 molar ratio and incubated at 37 ° c . for 2 hours . the mixture was then purified by a large cation exchange chromatography column ( resource s 200 - ml column , ge healthcare ) using sodium phosphate buffers ( buffer a : 25 mm na 2 hpo 4 , 25 % acetonitrile , ph 7 . 0 ), buffer b : 1 m nacl , 25 mm na 2 hpo 4 , 25 % acetonitrile , ph 7 . 0 ), a flow rate of 14 ml / min and a gradient of 0 - 100 % buffer b in 60 min , to remove unconjugated pmo and excess peptide . the conjugates were then desalted using 3 kda mwco amicon ultra - 15 centrifugal filter device ( millipore ) by concentring the sample ( 1 hour spin at 25 ° c ., at 3220 rcf ), then diluting the concentrate to 12 ml with h 2 o . the process was repeated twice . the conjugate was lyophilized and analysed by maldi - tof ms and hplc . peptide - pmo conjugates were dissolved in sterile water ( endotoxins free ) and filtered through 0 . 22 μm cellulose acetate membrane before use . the concentration was measured by uv at 265 nm in 0 . 1 n hcl using the extinction coefficient of the pmo as provided in the data sheet from gene tools . toxicity studies of pip7 - 9 - pmo were undertaken using cell viability assays . these showed that 10 arginine - pmo conjugates ( pip6e ) had a high level of cell toxicity ( fig4 ). therefore , employing pip 6a ( pip8 series ), pip6e ( pip7 series ) and pip6f ( pip9 series ) as parent peptides , pip7 - 9 - pmo series were synthesised by reducing the number of arginine and aminohexanoyl ( aminohexanoic acid ) residues in order to minimise possible cell toxicity . cell viability was substantially increased in pip7 - 9 - pmo treated mdx myotubes compared to pip6e treated cells ( fig4 ), suggesting promise for subsequent in vivo studies . a single intravenous injection of pip7 - 9 - pmo results in widespread dystrophin expression in the mdx skeletal / peripheral muscles to evaluate the exon skipping efficiency of pip7 - 9 - pmo compounds , 8 week old mdx mice were intravenously injected with 12 . 5 mg / kg of pip7 - 9 - pmo . at 2 weeks after injection , dystrophin expression was analysed in the tibialis anterior ( ta ), diaphragm , quadriceps and heart muscles . we evaluated the immune - stained dystrophin positive fibres , exon skipped mrna and dystrophin protein levels ( fig2 - 4 ). the intensity of dystrophin immuno - stained fibres was analysed using a rat anti - laminin antibody for normalisation and this was expressed as a ratio of the intensity level of c57bl / 10 muscles ( fig5 ). remarkable expression of exon - skipped dystrophin positive myofibres was observed in all pip7 - 9 - pmo injected skeletal muscles such as ta , quadriceps and diaphragm muscles at 2 weeks after injection ( fig4 - 46 ). however , only pip8b , pip8c , pip9b , pip9b2 and pip9c rendered significant dystrophin expression in the heart ( fig4 ). pip7a , pip7b , pip7b2 , pip8c2 , pip9d and pip9d2 resulted in negligible dystrophin production in the heart ( fig4 ). taking into account the structures of the peptides , it is apparent that the order of the amino acids in the hydrophobic core region is less important , as even the scrambled hydrophobic core of the pip9 series yielded similar dystrophin expression to the pip8 series in the heart . to evaluate the toxicity responses in pip7 - 9 - pmo treated mdx mice , we have undertaken preliminary evaluations of liver and renal function at 2 weeks after systemic administration of the pip7 - 9 - pmo compounds by extracting and analysing serum from treated mice and controls . analysis of serum creatine kinase ( ck ), creatinine , urea , alkaline phosphatase ( alp ), alanine aminotransferase ( alt ), aspartate aminotransferase ( ast ), total bilirubin , lactate dehydrogenase ( ldh ) levels was carried out . there was no elevation in the level of urea and creatinine , a marker for kidney toxicity and tbil , a marker for liver toxicity in ppmo treated mdx serum compared to untreated mdx control ( fig4 ). the alt , ast , alp and ldh levels were elevated in untreated mdx mice . there was no elevation in the levels of these markers in ppmo treated mdx serum compared to untreated mdx control . ck levels were variable between mice . we have identified ppmo to give high level of dystrophin restoration in both skeletal and heart muscle in mdx mice . pip8b , 9b , 9b2 , 8c and 9c have shown significant promise with very efficient dystrophin restoration in the tibialis anterior ( ta ), diaphragm , quadriceps and heart , without apparent toxicity . to compare exon skipping efficiency of lead ppmo , 8 week old mdx mice were intravenously injected with 12 . 5 mg / kg of ppmo . at 2 weeks after injection , dystrophin expression was analysed in the ta , quadriceps , diaphragm and heart muscles . we evaluated the immune - stained dystrophin positive fibres , exon skipped mrna and dystrophin protein levels ( fig4 - 50 ). the intensity of dystrophin immune - stained fibres in the ta and heart was analysed using a rat anti - laminin antibody for normalisation and this was expressed as a ratio of the intensity level of c57bl / 10 muscles ( fig4 ). levels of exon - skipped dystrophin rna and protein was remarkably expressed in pip8b , 9b and pip9b2 - pmo injected skeletal muscles such as ta , quadriceps , diaphragm and heart muscles at 2 weeks after injection ( fig4 - 50 ). however , pip8c and 9c - pmo rendered reduced dystrophin expression compared to pip8b , 9b and 9b2 - pmo . moreover , pip9d2 - pmo resulted in negligible dystrophin production in ta , quadriceps , diaphragm and heart muscles ( fig4 - 50 ), illustrating the importance of maintaining the length of the hydrophobic region of the peptide to induce efficient exon skipping . the dystrophin protein level in ppmo treated mdx muscles was quantified by western blot analysis . the density of the dystrophin band was quantified using licor software and vinculin was used as the loading control . the band density was normalised by its respective vinculin band and this was expressed as a percentage of the dystrophin expression level of c57bl / 10 muscles . the level of exon - skipped dystrophin protein was similar in pip8b , 9b and 9b2 - pmo injected skeletal muscles such as ta , quadriceps and diaphragm muscles at 2 weeks after injection ( fig5 ). however , pip8c and 9c rendered reduced dystrophin expression compared to 8b , 9b and 9b2 - pmo . moreover , 9d2 - pmo resulted in negligible dystrophin production in ta , quadriceps , diaphragm and heart muscles ( fig5 ). restoration of dysregulated microrna levels in lead ppmo ( exon23 mouse ) treated mdx mice we tested the ability of exon skipped dystrophin induced by ppmo candidates to correct microrna levels by ameliorating the muscle pathology in the mdx mice . we evaluated the levels of dystromirs ( micrornas dysregulated in dystrophin deficiency ), mir - 1 and mir - 133a , which were increased in mdx serum and were either decreased or unchanged in the mdx tissues and these may therefore constitute useful serum markers . at 2 weeks after intravenous injection of 12 . 5 mg / kg ppmo ( pip8b , 9b , 9b2 , 8c and 9c ) in 8 week old mdx mice , levels of mir - 133a and mir - 206 were examined by quantitative ( q )- pcr in ppmo treated serum and normalised to mir - 223 as a reference microrna . levels of mir - 133a and mir - 206 were significantly down - regulated in ppmo treated serum of mdx mice compared to untreated mdx controls , implying that muscle pathology was improved by ppmo ( fig5 ). to compare exon skipping efficiency of hplc and filter - desalted 9b2 - pmo , 8 week old mdx mice were intravenously injected with 12 . 5 mg / kg of ppmo . at 2 weeks after injection , dystrophin expression was analysed in the ta , quadriceps , diaphragm and heart muscles . we evaluated the immune - stained dystrophin positive fibres , exon skipped mrna and dystrophin protein levels ( fig5 - 54 ). the intensity of dystrophin immune - stained fibres was analysed using a rat anti - laminin antibody for normalisation and this was expressed as a ratio of the intensity level of c57bl / 10 muscles ( fig5 ). there was no change in the exon - skipped dystrophin intensity level at the sarcolemma induced by hplc and filter - desalted 9b2 - pmo . levels of exon - skipped dystrophin rna and protein was slightly increased in filter - desalted pip9b2 - pmo injected skeletal muscles compared to hplc - filtered pmo treated muscles , such as ta , quadriceps and diaphragm at 2 weeks after injection ( fig5 ). toxicity study of hplc and filter - desalted pip9b2 - pmo was undertaken using cell viability assays . cell viability was slightly increased in filter - desalted pip9b2 - pmo treated mdx myotubes compared to hplc - desalted pip9b2 - pmo treated cells , when they were treated with 60 μm of pip9b2 - pmo ( fig5 ). ppmo candidates have been identified to give high level dystrophin restoration in both skeletal and heart muscle in mdx mice . in particular , pip8b and 9b and pip9b2 , 8c and 9c have shown efficient dystrophin restoration in the tibialis anterior ( ta ), diaphragm , quadriceps and heart , without apparent toxicity . for the quantitative evaluation of dmd treatment in vivo , levels of dystromirs ( mir - 133a and mir - 206 ) have been evaluated which were increased in mdx serum . levels of mir - 133a and mir - 206 were significantly down - regulated in ppmo treated serums of mdx mice compared to untreated mdx control . pip7 and pip8 series peptides were tested in a cell viability assay , as follows . huh - 7 liver cells were grown at 37 ° c . under 5 % co 2 / 95 % air atmosphere in dmem supplemented with 10 % fetal bovine serum and penicillin / streptomycin antibiotics . cells were treated with trypsin and plated at 1 . 5 × 10 4 cells per well in 96 well plates . after overnight incubation cells were washed with pbs and pmo - peptide conjugates in 50 μl optimem were added to the wells in triplicate . after 4 hours incubation , conjugates were removed by replacement of media with 100 μl dmem / 10 % fbs for a further 20 h incubation . 20 μl of mts cell viability assay ( promega ) solution was added to the wells containing 100 μl dmem and plates were incubated for 1 - 2 hours before the uv measurements at 490 nm were taken . the cell viability percentage was determined by normalizing the average absorbance of triplicate samples to the mean of untreated samples . results presented ( fig5 and 58 ) are consistent with increased cell viability ( i . e . lower cellular toxicity ) of peptides containing fewer arginine residues . for example , peptides having a total of 7 arginine residues in domains 1 - 3 combined exhibited higher cell viability ( lower cellular toxicity ) than peptides having 8 or 9 arginine residues . in vitro assays : exon skipping in mdx mouse myotubes ; pip7 , pip8 and pip9 series h2k mdx myotubes were prepared and incubated with peptide - pmo conjugates in the absence of any transfection agent at concentrations of 0 . 125 , 0 . 25 , 0 . 5 , 1 . 0 and 2 . 0 μm by the method described previously [ yin , h ., et al ., pip 5 transduction peptides direct high efficiency oligonucleotide - mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice . mol ther , 2011 . 19 ( 7 ): p . 1295 - 303 .]. the products of nested rt - pcr from total isolated rna were examined by electrophoresis on a 2 % agarose gel . quantification of δ23 transcript levels was calculated using densitometry . the results ( fig5 to 61 ) show that pip7a , pip7b , pip7b2 , pip7c , pip7c2 , pip8a , pip8b , pip8c , pip8c2 , pip8d , pip9b , pip9b2 , pip9c , pip9c2 , pip9c3 , pip9d and pip9d2 pmo conjugates gave high exon skipping activity in mdx muscle cells . 2 . eckstein f . : expert opin . biol . ther . 2007 , 7 , 1021 . 3 . egholm m ., buchardt o ., nielsen p . e ., berg r . h . : j . amer . chem . soc . 1992 , 114 , 1895 . 4 . summerton j ., weller d . : antisense nucl . acid drug dev . 1997 , 7 , 187 . 5 . lebleu b ., moulton h . m ., abes r ., ivanova g . d ., abes s ., stein d . a ., iversen p . l ., arzumanov a ., gait m . j . : adv . drug delivery rev . 2008 , 60 , 517 . 6 . zatsepin t . s ., turner j . j ., oretskaya t . s ., gait m . j . : curr . pharm . design 2005 , 11 , 3639 . 7 . abes r ., arzumanov a ., moulton h . m ., abes s ., ivanova g . d ., iversen p . l ., gait m . j ., lebleu b . : biochem . soc . trans . 2007 , 35 , 775 . 8 . turner j . j ., arzumanov a ., ivanova g ., fabani m ., gait m . j . : cell - 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