One-year Treatment of Morpholino Antisense Oligomer Improves Skeletal and Cardiac Muscle Functions in Dystrophic mdx Mice

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1 originl rtile The Amerin Soiety of Gene & Cell Therpy One-yer Tretment of Morpholino Antisense Oligomer Improves Skeletl nd Crdi Musle Funtions in Dystrophi mdx Mie Bo Wu 1, Bin Xio 2,3, Cryn Cloer 1, Mon Shn 1, Arpn Sli 4, Peijun Lu 1, Jun Li 2, Knneoyin Ngrju 4, Xio Xio 2 nd Qi Long Lu 1 1 MColl-Lokwood Lortory for Musulr Dystrophy Reserh, Neuromusulr/ALS Center, Deprtment of Neurology, Crolins Medil Center, Chrlotte, North Crolin, USA; 2 Division of Moleulr Phrmeutis, Eshelmn Shool of Phrmy, University of North Crolin, Chpel Hill, North Crolin, USA; 3 The Institute of Hypertension, Deprtment of Internl Mediine, Tongji Hospitl, Tongji Medil College, Huzhong University of Siene nd Tehnology, Wuhn, People s Repuli of Chin; 4 Reserh Center for Geneti Mediine, Children s Ntionl Medil Center, Wshington, Distrit of Columi, USA Antisense therpy hs een suessful to skip trgeted dystrophin exon with orretion of frmeshift nd nonsense muttions of Duhenne musulr dystrophy (DMD). Systemi prodution of trunted ut funtionl dystrophin proteins hs een hieved in niml models. Furthermore, phse I/II linil trils in United Kingdom nd the Netherlnds hve demonstrted dystrophin indution y lol nd systemi dministrtions of ntisense oligomers. However, long-term effiy nd potentil toxiity remin to e determined. The present study exmined 1-yer systemi effet of phosphorodimidte morpholino oligomers (PMO) tretment trgeting mutted dystrophin exon 23 in mdx mie. PMO indued dystrophin expression dose-dependently nd signifintly improved skeletl musle pthology nd funtion with redued retine kinse (CK) levels y regimen of 6 mg/kg iweekly dministrtion. This regimen indued <2% dystrophin expression in the hert, ut improved rdi funtions demonstrted y hemodynmis nlysis. The results suggest tht low levels of dystrophin indution my e le to provide detetle enefit to rdi musle with limited myopthy. Body weight, serum enzyme tests, nd histology nlysis showed no sign of toxiity in the mie treted with up to PMO for 6 months. These results indite tht PMO ould e used sfely s effetive drugs for long-term systemi tretment of DMD. Reeived 14 Septemer 21; epted 29 Novemer 21; pulished online 21 Deemer 21. doi:1.138/mt Introdution Muttions in the dystrophin gene use two forms of musulr dystrophy, Duhenne nd Beker musulr dystrophy (DMD nd BMD). DMD is the most ommon lethl musle disorder minly resulting from nonsense nd frmeshift muttions preventing prodution of funtionl dystrophin protein. There is no effetive tretment ville. BMD is used y in-frme muttions tht typilly rete shortened, ut prtilly funtionl dystrophin, leding to vrile nd often overt symptoms. 1 3 The vriility of disese phenotype mong BMD popultion is the result of multiple ftors ffeting musle funtions. One of them is the funtionlity of the trunted dystrophin due to muttions involving different region nd the size of deletion. Suffiient preservtion of funtions y trunted (BMD-like) dystrophin underlies the priniple of reominnt deno-ssoited virus-medited minind mirodystrophin gene therpy. 4 The sme priniple hs now een utilized for the ntisense oligomer-medited exon skipping (ntisense therpy). In ntisense therpy, oligomers re speifilly designed to ind trget pre-mrna sequene for exlusion of trgeted exon(s). The removl of trgeted exon(s) restores dystrophin reding frme, resulting in the expression of BMD-like dystrophins. Fortuntely, most DMD muttions our within the rod domin of the dystrophin gene whih spns more thn hlf the length of the protein, ut seems to hve limited funtionl importne. 5,6 Antisense therpy therefore will e le to remove the mutted or dditionl exon(s) tht disrupt the reding frme, thus restoring the expression of shortened forms of dystrophin protein retining ritil funtions in most DMD ptients. Lst 1 yers hve witnessed the signifint progress in ntisense therpy for DMD Erly studies demonstrted the fesiility of ntisense oligonuleotides (AO) to remove trgeted dystrophin exon in mouse nd humn ells. 7,8,1 In 23, AO ws for the first time shown to e le to restore funtionl levels of dystrophin in the dystrophi mdx mie y intrmusulr injetions. 12 Adeno-ssoited virus-medited prodution of AO hs een shown to e highly effetive for trgeted exon skipping oth lolly nd systemilly with long-term therpeuti effet. 6 This pproh thus hs strong dvntge over syntheti AOs y eliminting the need for repeted injetions. However, estlishing the Correspondene: Qi Long Lu, MColl-Lokwood Lortory for Musulr Dystrophy Reserh, Neuromusulr/ALS Center, Deprtment of Neurology, Crolins Medil Center, 1 Blythe Blvd. Chrlotte, North Crolin 28231, USA. E-mil: qi.lu@rolinshelthre.org or Bo Wu, MColl-Lokwood Lortory for Musulr Dystrophy Reserh, Neuromusulr/ALS Center, Deprtment of Neurology, Crolins Medil Center, 1 Blythe Blvd. Chrlotte, North Crolin 28231, USA. E-mil: o.wu@rolinshelthre vol. 19 no. 3, mr. 211

2 The Amerin Soiety of Gene & Cell Therpy One-yer Morpholino Improves Musle Funtion in mdx Mie effiy of deno-ssoited virus-medited gene delivery systemilly in humn will likely tke prolonged period. In ontrst, the effet of syntheti AOs on exon skipping is short-lived nd short-term toxiity of most widely used AO hemistries hs een estlished. This provides ses for regultory uthorities to pprove syntheti AOs for linil trils more willingly. As result, linil trils trgeting humn dystrophin exon 51 hve een onduted in the Europe to test the priniple for treting sugroup of DMD popultion. 19,2 Sine then, AO-medited exon skipping with syntheti AOs hs progressed into one of the most promising experimentl therpy for DMD. Systemi effiy with funtionl improvement hs een demonstrted in oth dystrophi mouse nd dog models. 13,14,18 The use of peptides nd tioni polymers for enhned delivery of phosphorodimidte morpholino oligomers (PMO) hs hieved restortion of dystrophin expression to ner norml levels in ody-wide skeletl musles nd to less degree in the rdi musle. 15,16 High level expression of the restored dystrophin improved funtions of oth dystrophi skeletl nd rdi musles signifintly. However, inresed toxiity nd potentil immune response to the tioni peptide nd polymers demnd more thorough prelinil tests efore these modified AOs n e triled in linis. Therefore, linil trils for exon skipping re urrently eing limited to the use of unmodified PMO nd 2 -O-methyl phosphorothiote (2OMePS) hemistries. Completed phse I linil trils y lol musle injetions trgeting exon 51 reported the signifint mount of trgeted exon skipping nd dystrophin prodution in nerly ll treted musles with oth 2OMePS nd PMO. 19,2 More reently, similr to tht oserved in the niml models, systemi tretment of DMD ptients with PMO ws reported to indue detetle mount of dystrophin protein in smpled skeletl musles of ll ptients, lthough onsiderle vrition in skipping effiieny ws lso lerly oserved within the tril popultion. 21 AO drugs t on pre-mrna of dystrophin for the removl of trgeted exon. The therpeuti effet is therefore short-lived with single dministrtion. Previous investigtions suggested tht the effet of dystrophin indution fter single intrvenous (i.v.) injetion of PMO in mdx mie lsted ~2 months. 12 Antisense therpy for DMD requires life-long dministrtion nd the pity of the drugs to mintin funtionl levels of dystrophin with tolerle toxiity. It is therefore importnt to understnd whether therpeuti levels of dystrophin n e mintined y regulr injetions for prolonged time. However, nerly ll studies of i.v. AO dministrtions in niml models hve so fr limited to short-time period. It is espeilly importnt s delivery effiieny of oth ntisense hemistries, 2OMePS nd PMO, is pprently relted to the stte nd degree of musle degenertion nd regenertion, eing less effetive in norml musles when ompred to degenertive musles. This rises the onern tht regulr injetions of AO, espeilly t low dosge might not e le to hlt the yles of degenertion when only limited mount of dystrophin is produed in only proportion of musle fiers. Effiieny of exon skipping nd dystrophin prodution in rdi musle with unmodified 2OMePS nd PMO is signifintly lower thn tht in skeletl musles. This lso uses onern tht restortion of higher levels of dystrophin in skeletl musles my exerte the filure of hert funtion if dystrophin expression nnot e effetively restored in rdi musle. 22 In the present study, we investigted long-term dosge effet of PMO tretment in the dystrophi mdx mie. We demonstrted tht PMO ws le to indue effetive trgeted exon skipping nd dystrophin expression in ll musles dose-dependently during 1-yer tretment lthough umultive effet ws limited. Higher dosges of PMO were ssoited with inresing levels of dystrophin expression nd improvement of musle pthology nd funtions without detetle toxiity. Results One-yer i.v. dministrtion of 15 mg/kg PMO reveled no signifint improvement in musle pthology We hve erlier shown tht single i.v. injetion of 15 mg/kg PMO trgeting mouse dystrophin exon 23 ws le to indue lower, ut detetle levels of dystrophin expression. 17 As the hlf-life of PMO-indued dystrophin expression ws reported to e out 2 months, we hypothesized tht repeted dministrtion with this dose of PMO iweekly might led to higher levels of dystrophin indution, suffiient to hieve therpeuti signifine. To test this, we treted the mdx mie with 15 mg/kg PMO iweekly for 1 yer. Two weeks fter the lst injetions, skeletl nd rdi musles were exmined. The revertnt fiers of <1.5% were deteted in ll musles from ontrol mdx mie. Dystrophin positive fiers inresed to up to 5% in ll the skeletl musles from the PMOtreted mie, lthough smll numer of fiers with wek prtil dystrophin positive signl ws lso oserved (Figure 1). The mximum mount of dystrophin protein exmined y western lots ounted for 2.6% of norml level in ny skeletl musles (Figure 1). This effet ws therefore similr to tht oserved in musles fter single injetion of 15 mg/kg PMO, suggesting no signifint umultion in exon-skipping effet y the dosing regimen. No ler differene in pthology nd vrition in fier size of skeletl musle were oserved (Figures 1e nd 2). Sirius stining showed no ler redution in stining intensity for ollgens in the treted musles when ompred to the ontrols (Supplementry Figure S1). Consistently, the funtion of the skeletl musles mesured y grip-fore genertion were only mrginlly improved with no differene in retine kinse (CK) levels in the treted group when ompred to the untreted ontrol mdx mie (Figure 2,). No dystrophin expression nd speifi exon 23 skipping were deteted in the rdi musle nd mesurement for rdi funtions reveled no signifint differene from the untreted ontrol mdx mie (Figures 1 nd 3; Tles 1 nd 2). Body weight, serum enzyme tests, nd histologil exmintion of lung, kidney, nd liver showed no differenes etween the treted nd the untreted ontrol mdx mie (Figure 2). One-yer i.v. dministrtion of 6 mg/kg PMO signifintly improved skeletl musle pthology Erlier study showed tht single i.v. injetion of 2 mg PMO per dult mdx mie (~6 8 mg/kg) ws le to indue dystrophin expression in ody-wide skeletl musles of mdx mie. 14 We therefore exmined long-term effet of 6 mg/kg PMO y regulr iweekly i.v. injetions. Skeletl musles fter 1-yer tretment sw Moleulr Therpy vol. 19 no. 3 mr

3 The Amerin Soiety of Gene & Cell Therpy One-yer Morpholino Improves Musle Funtion in mdx Mie C57 15 mg/kg PMO 6 mg/kg PMO < ± 3.11 Figure 1 Restortion of dystrophin expression fter 1-yer intrvenous (i.v.) tretment of 15 or 6 mg/kg phosphorodimidte morpholino oligomers (PMO). () Detetion of dystrophin y immunohistohemistry with rit polylonl ntiody P7 ginst dystrophin. Blue nuler stining with DAPI. () Detetion of exon 23 skipping in musles y reverse trnsription-pcr (RT-PCR). Lne 1 size mrker. () Western lot showed levels of dystrophin expression in musles [1 µg totl protein loded from tiilis nterior () musle of C57BL/6 mouse; 5 µg totl protein loded from untreted or PMO-treted mdx mie]. C57-, musle from norml C57BL/6 mouse; Cont-, musle from untreted mdx mouse. 15 or 6 mg/kg, the musles from 15 or 6 mg/kg PMO-treted mdx mie. The smples from two different mdx mie treted with 6 mg/kg PMO were shown. (d) α-atin s loding ontrols. (e) The perentge of non-entrnuleted musle fiers., musles from untreted mdx mouse. (n = 1; P.5 ompred with untreted mdx mie. Two-tiled t-test). signifint indution of trgeted exon 23 skipping nd dystrophin expression. The levels of exon 23 skipping y reverse trnsriptionpcr (RT-PCR) rnged from rely detetle to 4% in skeletl musles (Figure 1). Dystrophin positive fiers, with highly vrile intensity, were ounted from 1 to 5% in skeletl musles exmined (Figure 1). Suh vrition ws seen etween different musles s well s within the sme type of musles from different mdx mie. Vrition in exon-skipping effiieny ws lso demonstrted y western lots with the levels of dystrophin protein up to 17.1% (Figure 1). The treted mie showed lerly detetle improvement in skeletl musle pthology with redued entrl nuletion (Figure 1e) nd more homogenous popultion of /l) l) G G T (U /d l) AL P (U l) /d AL T u ilir 8 6 C57 15 mg/kg PMO 6 mg/kg PMO 4 2 Forelim Hindlim To t l t (U g/ e (K ± 2.1 in se 6 mg/kg PMO ± 1.73 re ki e in t re Gstronemius Bieps 9.29 ± ± ± ± ± ± C Hert Diphrgm Bieps Hert Diphrgm 6 mg/kg C n % 15 mg/kg PMO Qudrieps Bieps Hert Diphrgm Qudrieps Cont- C57-15 mg/kg 578 (m U d e l) g/ (m 1. >1 /l) <1 <1 in % Avg. mx. strength (KGF) Figure 2 Exmintion of pthology, serum, nd skeletl musle funtion fter 1-yer tretment of 15 nd 6 mg/kg phosphorodimidte morpholino oligomers (PMO). () Histology [hemtoxylin nd eosin (H&E) stining] of tiilis nterior (), diphrgm, liver, nd kidney from the norml C57BL/6 mie, untreted mdx mie (ontrol), 15 or 6 mg/kg PMO-treted mdx mie (15 mg/kg, 6 mg/ kg). () The levels of serum enzymes. Cretine kinse (KU/l), retinine (mg/l), ure nitrogen (mg/ml), totl iliruin (mg/l), diret iliruin (mg/dl), lnine trnsminse (ALT) (U/dl), lkline phosphtse (ALP) (U/dl), nd γ-glutmyltrnsferse (GGT) (U/l). A signifint redution in retine kinse levels ws oserved in mdx mie treted with 6 mg/kg PMO when ompred with untreted mdx mie. () Grip strength mesurement. Signifint improvement ws oserved in the mie fter tretment with 6 mg/kg PMO. KGF, kilogrm fore. (n = 1; P.5 ompred with C57BL/6 mie; P.5 ompred with untreted mdx mie. Two-tiled t-test). Finl men ody weight: C57BL/6, 31.3 ±.8 g; untreted mdx, 34.1 ± 2.; 15 mg/kg PMOtreted mdx, 31.8 ± 2.4 g; 6 mg/kg PMO-treted mdx, 33.7 ± 1.6 g. PMO, phosphorodimidte morpholino oligomers. fiers in size (Figure 2). Sirius stining showed ler redution in the intensity for ollgens in the treted skeletl musles when ompred to the ontrols (Supplementry Figure S1). CK levels in the treted mie were signifintly redued when ompred to the untreted ontrol mdx mie, ut remined higher thn tht of norml C57BL/6 mie (Figure 2). Signifint improvement ws lso deteted in musle funtions with grip-fore genertion (Figure 2). The results suggest tht this dosge of PMO tretment n hieve mesurle therpeuti outome in skeletl musles without detetle toxiity. Improvement in rdi funtions fter 1-yer tretment of 6 mg/kg PMO The effiieny of exon skipping in the rdi musle ws onsiderly lower thn tht in skeletl musles fter the 1-yer PMO tretment. Less thn 5% dystrophin positive fiers, most of them with disontinuous wek signls t the ell memrne, were deteted in vol. 19 no. 3 mr. 211

4 The Amerin Soiety of Gene & Cell Therpy One-yer Morpholino Improves Musle Funtion in mdx Mie e Stroke volume (RVU) End systoli pressure (mm Hg) dp/dt mx (mm Hg/se) g dv/dt mx (RVU/se) , 7,5 5, 2, Crdi output (RVU/min) 3, 2, 1, the rdi musle (Figure 1). The levels of dystrophin protein were only up to 2% y western lot in ll hert of treted mie (Figure 1). To determine the potentil effet of the tretment with suh limited protein indution on rdi funtions, we exmined the rdi musles first y ultr-high frequeny ehordiogrphy. No signifint differene in histology nd ventriulr funtion of the hert ws identified etween the treted group nd the untreted ontrol mie. We further exmined the hert funtions of the PMO-treted mdx mie y hemodynmis nlysis under doutmine stress in omprison with the untreted ontrol mdx mie nd norml C57BL/6 mie. This nlysis hs een reported to e highly sensitive to distinguish the degree of underline funtionl defets nd to demonstrte potentil therpeuti effet in the rdi musle of the mdx mie. 15 The untreted ontrol mdx mie showed ler defiits of rdi funtions with redution in the stoke volume, rdi output, mximum upstroke veloity (dv/ dt mx ), ventriulr isovolumi relxtion time onstnt (τ), ejetion frtion, nd inrese of minimum upstroke veloity (dv/dt min ), when ompred to the norml C57BL/6 mie (Tle 1). One-yer d f dp/dt min (mm Hg/se) Ejetion frtion (%) dv/dt min (RVU/se) , 4, 6, 8, Figure 3 Crdi funtions in the mdx mie treted with 15 or 6 mg/kg phosphorodimidte morpholino oligomers (PMO) (1 yer) y hemodynmis t 15 minutes fter doutmine hllenges. Signifint improvement ws oserved in () stroke volume, () rdi output, () end-systoli pressure, (d) ejetion frtion, (e) dp/dt mx, (f) dp/dt min, (g) dv/dt mx, (h) dv/dt min. (1) Age-mthed norml C57BL/6 mie; (2) ge-mthed untreted mdx mie; (3) 1-yer 15 mg/kg PMOtreted mdx mie; (4) 1-yer 6 mg/kg PMO-treted mdx mie. (n = 1; P.5 in omprison with C57BL/6 mie; P.5 in omprison with untreted mdx mie. Two-tiled t-test). h tretment of 6 mg/kg PMO slightly improved the stoke volume, rdi output, ventriulr isovolumi relxtion time onstnt (τ), nd ejetion frtion, ut the hnges were not sttistilly signifint. Signifint improvement ws however, reveled in the mximum nd minimum upstroke veloity (dv/dt mx nd dv/dt min ) (Tle 1). Under doutmine stress, the untreted ontrol mdx mie showed signifint rdi dysfuntions, inluding dereses in stoke volume, rdi output, end-distoli volume, end-systoli pressure, dp/dt mx, dv/dt mx, ejetion frtion, nd n inrese in dp/dt min, dv/dt min (Figure 3 nd Tle 2). Under the sme stress ondition, the 6 mg/kg PMO-treted mdx mie showed improved rdi funtions, espeilly in stoke volume, rdi output, enddistoli volume, end-systoli pressure, dp/dt mx, dv/dt mx, dv/ dt min, nd ejetion frtion (Figure 3 nd Tle 2). Repeted high-dose tretments further enhned the exon-skipping effiieny nd skeletl musle funtion The dose-dependent short- nd long-term exon-skipping effet nd funtionl improvement prompted us to exmine the potentil of PMO tretment with two higher doses of 3 mg/kg nd 1.5 g/ kg. Six months of iweekly tretment with PMO t 3 mg/kg were le to hieve dystrophin expression in >5% fiers in ody-wide skeletl musles (Figure 4). The levels of trunted dystrophin mrna rnged from 25 to 4 % (Figure 4). Western lot deteted >15% of norml levels of dystrophin protein in ll skeletl musles (Figure 4). Musle pthology ws lerly improved with none to only few individul degenerting fiers deteted in ny skeletl musles exmined (Figure 5). The fiers within the sme musle eme highly uniform nd with no ler inflmmtory ell infiltrtions. Sirius stining showed very ler redution in the intensity for ollgens in the treted skeletl musles when ompred to the ontrols nd the lower dose-treted mdx mie (Supplementry Figure S1). Consistently, the CK levels were signifintly redued when ompred to those untreted ontrol mie nd the mie with lower doses of PMO tretment (Figures 2 nd 5). Musle funtions s mesured y grip-fore genertion were signifintly improved (Figure 5). Dystrophin expression with strong signl ws lso deteted in smll proportion of rdi musle fiers nd the mount of dystrophin protein eme detetle lthough still <4% in ll hert of the treted mie (Figure 4). Inresing the dose of PMO to sw further improvement of dystrophin indution in ll musles. Nerly 1% fiers in ll skeletl musles expressed dystrophin y immunohistohemistry with the levels of the protein rehing 25 5% (Figure 4,). This level of dystrophin expression ws ssoited with signifint improvement in musle funtion with the grip fore rehing the strength ner to the norml C57BL/6 mie (Figure 5). CK levels redued drmtilly ompred to those untreted ontrol mie (Figure 5). All skeletl musle fiers eme highly uniform in size, nd degenerting fiers nd inflmmtory infiltrtes were sent in ll musles (Figure 5). More thn 4% of rdi musle fiers expressed dystrophin lthough most of them hd wek signl for the protein. During the 6-month tretment with these two high dosges, the mie showed no sign of norml ehvior nd hnge in ody weight. No pthologil hnge of the liver, kidney, nd lung ws oserved y hemtoxylin nd eosin stining Moleulr Therpy vol. 19 no. 3 mr

5 One-yer Morpholino Improves Musle Funtion in mdx Mie The Amerin Soiety of Gene & Cell Therpy Tle 1 Bseline hemodynmi dt C57 15 mg/kg PMO 6 mg/kg PMO Hert rte (pm) 45.3 ± ± ± ± 18. Stoke volume (RVU) 4.7 ±.4 4. ±..2 ± ±.6 Crdi output (RVU/min) 1,99.4 ± ,6.9 ± ,536.8 ± ,938.8 ± 31.8 End-systoli volume (RVU) 13.8 ± ± ± ±.8 End-distoli volume (RVU) 17.4 ± ± ± ±.8 End-systoli pressure (mm Hg) 9. ± ± ± ± 4.6 End-distoli pressure (mm Hg) 7.5 ± ± ± ± 1.9 Arteril elstne (mm Hg/RVU) 2.6 ± 2..7 ± ± 2..7 ± 2.8 dp/dt mx (mm Hg/se) 7,87.6 ± ,815.3 ± ,828.2 ± 1,139. 7,66. ± dp/dt min (mm Hg/se) 7,66.1 ± ,291. ± ,775.5 ± 1,41.7 6,947.2 ± dv/dt mx (RVU/se) ± ± ± ± 6.4 dv/dt min (RVU/se) 22.2 ± ± ± ± 28.9 τ (ms) 9.5 ± ± ± ± 1.1 Ejetion frtion (%) 26.5 ± ± ± ± 2.4 Arevitions: 15 mg/kg PMO, 1-yer 15 mg/kg PMO-treted mdx mie; 6 mg/kg PMO, 1-yer 6 mg/kg PMO-treted mdx mie; C57, norml C57BL/6 mie; ontrol, untreted mdx mie; PMO, phosphorodimidte morpholino oligomers; RVU, reltive volume units. n = 1; P.5 in omprison with C57BL/6 mie; P.5 in omprison with untreted mdx mie. Two-tiled t-test. Tle 2 Hemodynmi prmeters 15 minutes fter doutmine infusion C57 15 mg/kg PMO 6 mg/kg PMO Hert rte (pm) 41.1 ± ± ± ± 7. Stoke volume (RVU) 5.4 ± ± ± ±.9 Crdi output (RVU/min) 2,135.2 ± ,332.2 ± ,428.2 ± ,211. ± End-systoli volume (RVU) 13.4 ± ± ± ±.8 End-distoli volume (RVU) 18.8 ± ± ± ± 2. End-systoli pressure (mm Hg) 88.4 ± ± ± ± 4.3 End-distoli pressure (mm Hg) 6.7 ± ± ± ± 1.9 dp/dt mx (mm Hg/se) 8,659.9 ± ,2.3 ± ,627. ± 1,53.8 7,95.2 ± 49.8 dp/dt min (mm Hg/se) 7,212.7 ± ,111.1 ± ,713. ± ,741. ± dv/dt mx (RVU/se) ± ± ± ± 32.6 dv/dt min (RVU/se) 24. ± ± ± ± 21.4 τ (ms) 8.2 ±.7 8. ± ± ±.9 Ejetion frtion (%) 29.9 ± ± ± ± 3.9 Arevitions: 15 mg/kg PMO, 1-yer 15 mg/kg PMO-treted mdx mie; 6 mg/kg PMO, 1-yer 6 mg/kg PMO-treted mdx mie; C57, norml C57BL/6 mie; ontrol, untreted mdx mie; PMO, phosphorodimidte morpholino oligomers; RVU, reltive volume units. n = 1; P.5 in omprison with C57BL/6 mie; P.5 in omprison with untreted mdx mie. Two-tiled t-test. nd serum tests for ure nitrogen, totl iliruin, diret iliruin, lkline phosphtse, nd γ-glutmyltrnsferse. (Figure 5,). Disussion Sine the first demonstrtion of therpeuti effet in vivo in DMD niml model of mdx mie, AO-medited exon skipping hs progressed into one of the most promising experimentl therpies. Short-term systemi dministrtion of ntisense PMO is le to hieve funtionl levels of dystrophin expression in oth mouse nd dog models of DMD. 14,18 Dt from the ongoing linil tril of PMO trgeting exon 51 hs onfirmed systemilly indued dystrophin expression in musles of DMD oys fter weekly tretment. 21 However, effetive exon skipping for DMD requires life-long dministrtion nd the pity of ntisense drugs to mintin funtionl levels of dystrophin with tolerle toxiity. In this study, we demonstrted tht systemilly dministrted AO s PMO ws indeed le to indue effetive trgeted exon skipping nd dystrophin expression in ll musles in dose- dependent mnner for 1 yer in mdx mie. Bodyweight, pperne, nd ehvior of the treted niml with ll dosges were not signifintly different from the ontrol groups during the period of 1-yer tretment. Serum tests nd histology nlysis showed no pthologil hnges in the liver, kidney nd lung. Lk of toxiity ws further demonstrted in the mie treted with up to 1.5 g/ kg PMO for 6 months. These results together suggest tht PMO tretment n hieve long-term therpeuti effets nd is well tolerted vol. 19 no. 3 mr. 211

6 The Amerin Soiety of Gene & Cell Therpy One-yer Morpholino Improves Musle Funtion in mdx Mie d C57- Cont- 3 mg/kg Approprite dosge of AO drug given systemilly is ritil for hieving long-term therpeuti effiy to DMD. Prt of the present study imed to ssess the potentil therpeuti dosge nd umultive effet with long-term regulr dministrtion of PMO. Our results showed tht trgeted exon skipping nd expression of dystrophin n e deteted t the lowest dose of 15 mg/kg with iweekly i.v. injetions lthough this dosge is pprently insuffiient to hieve signifint levels of dystrophin indution nd therpeuti effet. When 6 mg/ kg PMO ws given iweekly, exon 23 skipping nd dystrophin expression were lerly demonstrted in ll musles. Signifint improvement in musle pthology ws indited y redutions in musle dmge, entrl-nuleted fiers, ollgen deposit, nd serum CK levels. Furthermore, improvement in musle funtion ws reorded y grip-fore mesurement. This result suggests tht 6 mg/kg PMO ws le to hieve lerly detetle therpeuti effet with mesurle improvement in musle funtions in the dystrophi mdx mouse. The result is onsistent to our erly oservtion tht similr dose of PMO with 7 weekly tretments is le to hieve lerly detetle improvement in musle funtions. 14 Therefore, this dosge ould e onsidered s useful referene to tretment regimen iming to hieve effiy with PMO-medited exon skipping systemilly. 3 mg/kg Diphrgm 3 mg/kg Hert Figure 4 Restortion of dystrophin expression in the mdx mie fter 6 months intrvenous (i.v.) tretment with phosphorodimidte morpholino oligomers (PMO) (3 mg/kg or iweekly). () Detetion of dystrophin y immunohistohemistry with rit polylonl ntiody P7. Blue nuler stining with DAPI. () Detetion of exon 23 skipping in musles y reverse trnsription-pcr (RT-PCR). Lne 1, size mrker. () Western lot for dystrophin expression [25 µg totl protein loded from tiilis nterior () musle of C57BL/6 mouse; 5 µg totl protein loded from untreted or treted mdx mie]. C57-, musle from norml C57BL/6 mouse; Cont-, musle from untreted mdx mouse. 3 mg/kg, 3 mg/kg PMO-treted mdx mie. PMO, PMO-treted mdx mie. (d) α-atin s loding ontrols Cretine kinse (KU/l) Cretine (mg/l) C57 3 mg/kg Ure nitrogen (mg/ml) Totl iliruin (mg/l) ALT (U/dl) ALP (U/dl) GGT (U/l) However, ution needs to e exerised when the dt from this study re extrpolted for the purpose of the tretment of DMD. Effiy of ntisense therpy to tret individul DMD ptient, depends on mny other ftors, espeilly the effiieny of individul ntisense drug nd the funtionlity of the trunted dystrophin determined minly y the lotion of muttions nd size of deletions. Both determinnts re diffiult or yet to e defined for trgeting most humn dystrophin exons. However, the following two ftors re likely to ffet our onsidertion for the referene vlue of effetive dosge oserved in the mdx mie. First, it is most prole tht trunted dystrophin lking only exon 23 produed in the mdx mie musles will retin etter funtion thn most trunted dystrophins expeted from DMD ptients fter exon skipping. Seond, the PMOE used in the present study hs een optimized nd tested extensively with high degree effiieny in oth ell ulture nd in vivo. This is in ontrst to the lk of pproprite system to verify exon-skipping effiieny in vivo for AO drugs trgeting humn dystrophin exon. It is therefore likely tht onsiderle higher dosge my e required to hieve lerly mesurle therpeuti outome with most AO drugs trgeting humn dystrophin exons in lini providing tht dose effet is similr etween mouse nd humn on weight/weight Avg. mx. strength (KGF) C57 3 mg/kg Forelim Hindlim Figure 5 Exmintion of pthology, serum, nd skeletl musle funtion fter 6 months tretment with 3 mg/kg nd phosphorodimidte morpholino oligomers (PMO). () Histology [hemtoxylin nd eosin (H&E) stining] of tiilis nterior (), diphrgm, nd kidney from untreted (ontrol), 3 mg/kg nd PMO-treted mdx mie (3 mg/kg, ). () The levels of serum enzymes. Cretine kinse (KU/l), retinine (mg/l), ure nitrogen (mg/ml), totl iliruin (mg/l), diret iliruin (mg/dl), lnine trnsminse (ALT) (U/dl), lkline phosphtse (ALP) (U/dl), nd γ-glutmyltrnsferse (GGT) (U/l). () Grip strength tests. Signifint improvement ws oserved in the mdx mie treted with 3 mg/kg nd PMO. KGF, kilogrm fore. (n = 1; P.5 in omprison with C57BL/6 mie; P.5 in omprison with untreted mdx mie. Two-tiled t-test). Moleulr Therpy vol. 19 no. 3 mr

7 One-yer Morpholino Improves Musle Funtion in mdx Mie The Amerin Soiety of Gene & Cell Therpy sis. When the outome of the ongoing linil trils eomes ville, it might e possile to estlish reltive effiieny of new AO drugs trgeting different exons with the AO on tril s the ontrol. This n e hieved y testing two AOs together in the sme ell ulture nd/or in the sme niml model of humnized mdx mie. This together with our understnding of funtionlity of individul trunted dystrophin would provide more urte ssessment for dosing to hieve effiy in linis. DMD muttions ffet the expression of dystrophin in ll musles inluding rdi musle. While defets in rdi funtions s result of dystrophin defiieny is not prominent in the erly stge of disese mnifesttion, filures of rdi funtion, nd the respirtory system re the leding uses of lethlity in lter stge of disese progression. Thus, restortion of funtionl levels of dystrophin to prevent deteriortion in rdi funtions onstitutes one of the ritil ims for the exon skipping to e effetive therpy. Furthermore, s DMD ptients live longer due to improved multidisiplinry ptient re, resuing dystrophin expression in rdi musle eomes more ritil for their longevity nd qulity of life Erly studies however, reveled tht the effiieny of ntisense oligomer-indued exon skipping ws signifintly (severl folds) lower in rdi musle thn in skeletl musles. Systemi PMO tretment t the doses <3 mg/kg produed only rely detetle (<5% norml levels) dystrophin in the rdi musle. 14,17 This lower effiieny of exon skipping eomes one of the mjor onerns for the therpy to e effetive to DMD ptients. The results from the present study onfirmed tht the rdi musle is muh more resistnt to PMO-medited exon skipping. Only 2% or less norml levels of dystrophin expression were deteted in the hert fter 1-yer i.v. injetions of 6 mg/kg PMO. The result lso indites limited umultive exon-skipping effet in the rdi musle with the iweekly tretment regimen. Nevertheless, funtionl improvement, lthough limited, ws lerly demonstrted y the hemodynmis nlysis. This result suggests tht low levels of dystrophin indution my e le to provide enefit to rdi funtions lthough other ftors suh s improved funtions in skeletl musle espeilly the diphrgm ould lso e involved. 22 However, it is lso possile tht suh low levels of dystrophin indution, eing suffiient to provide improvement to the mildly ffeted rdi musle of the mdx mie, my not e suffiient to improve funtions of severely ffeted rdi musles in DMD ptients. The hlf-life of AO-indued dystrophin protein hs een estimted to e out 2 months lthough it is not ler whether this is the result of protein stility or the sustined effet of AO within the fiers. Previous studies oserved signifint umultion in dystrophin protein y short-term weekly or dily injetions. 14 Therefore, we expeted tht the levels of dystrophin fter 1-yer iweekly injetions would e signifintly higher thn tht fter single injetion when the sme dose of PMO is used. Unexpetedly, the results showed tht the long-term umultive effet of PMO ws limited. The mehnism(s) is not understood, ut the following ftors my e involved. Firstly, iweekly regimen will ertinly hve less umultive effet thn weekly regimen. Perhps more importntly, the dependene of unmodified PMO on the inresed memrne permeility for effetive delivery my onstitute nother possile explntion. Sine the effiieny of PMO relies hevily on permeility of musle fiers nd dystrophi musles undergo yles of degenertion nd regenertion, the initil few doses of PMO would hieve higher effiieny through the frtion of musle fiers with higher permeility. However, suh fier popultion dereses grdully nd most musle fiers eome less permele to PMO eventully, leding to diminishing delivery effiieny, thus umultive effet. This hypothesis is onsistent with the ft tht norml musles re resistnt to PMO delivery. 14 While the limited umultive effet my indite the improvement in musle memrne permeility fter the tretment, it lso suggests tht long-term effiieny in exon skipping with PMO nd 2OMePS my e diffiult to exeed tht hieved y single dose of AO. Mterils nd Methods Animls, oligonuleotides, nd in vivo delivery methods. Ten mdx mie nd C57BL/6 mie ged 4 5 weeks were used in eh group. Experiments were pproved y IACUC Crolins Medil Center (protool A for reeding of mdx mie; protool 1-8-7A for PMO oligonuleotide therpy for DMD). The PMO (+7-18) (5 -GGCCAAACCTCG GCTCCTGAAAT-3 ) ginst the oundry sequenes of exon nd intron 23 of dystrophin gene (PMO) were used (Gene Tools, Philomth, OR). For i.v. dministrtion, PMO ws used in 1-µl sline y retrooritl injetions. Sme volume of sline ws used in ontrol mie. Mie were killed t desired time points, nd musles were snp-frozen in liquid nitrogen-ooled isopentne nd stored t 8 C. Antiodies nd immunohistohemistry. Setions of 6 µm were ut from t lest two-thirds of musle length of tiilis nterior, qudrieps, ieps, nd gstronemius t 1 µm intervls nd t lest 1 levels from ll other musles inluding hert, diphrgm, interostls, nd dominl musles t 1 µm intervls. The intervening musle setions were olleted either for western lot nd RT-PCR nlysis. The seril setions were stined with rit polylonl ntiody P7 ginst dystrophin sequenes from mino id 2847 to mino id The primry ntiody ws deteted y got-nti-rit immunogloulin Gs Alex 594 (Invitrogen, Eugene, OR). Setions were lso stined with hemtoxylin nd eosin or pirosirius red solution for histologil nd ollgen ssessment. Protein extrtion nd western lot. The olleted setions were ground into powder nd lysed with 2-µl protein extrtion uffer s desried previously. 15,16 The protein onentrtion ws quntified y Protein Assy Kit (Bio-Rd, Herules, CA). Proteins were loded onto 4 15% Tris HCL grdient gel. Smples were eletrophoresed overnight t 1 ma t 4 C nd lotted onto nitroellulose memrne overnight t 5 V. The memrne ws then wshed nd loked with 5% skimmed milk nd proed with monolonl ntiody NCL-DYS1 ginst dystrophin rod domin (Vetor Ls, Burlingme, CA) overnight. The ound primry ntiody ws deteted y horserdish peroxidse-onjugted got nti-mouse immunogloulin G nd ECL Western Blotting Anlysis System (Perkin Elmer, Wlthm, MA). The intensity of the nds otined from the oligomertreted mie musles ws mesured nd ompred with tht from norml musles of C57BL/6 mie (NIH ImgeJ 1.42 softwre). RNA extrtion nd RT-PCR. The olleted setions were homogenized in TRIzol (Invitrogen) y using n Ultr-Turrx homogenizer (Jnke nd Kunkel, Stufen, Germny). Totl RNA ws then extrted nd 1 ng of RNA templte ws used for 5-ml RT-PCR with RT-PCR Mster Mix (USB, Clevelnd,OH). The primer sequenes for the RT-PCR were Ex2Fo 5 -AGAATTCTGCCAATTGCTGAG-3 nd Ex26Ro 5 -TTCTTCAGCT TGTGTCATCC-3 for mplifition of mrna from exons 2 to 26. A totl of 4 yles were rried out for the RT-PCR. Bnds with the expeted size for the trnsript with exon 23 deleted were extrted nd sequened. The intensity of the nds ws mesured with the NIH ImgeJ 1.42 nd perentge of exon skipping ws lulted with the intensity of the two nds representing oth exon 23 unskipped nd skipped s 1% vol. 19 no. 3 mr. 211

8 The Amerin Soiety of Gene & Cell Therpy One-yer Morpholino Improves Musle Funtion in mdx Mie Crdi imging. Mie were pled in n indution hmer with 4% isoflurne mixed with 1% oxygen. The mouse ws monitored for hert rte (35 55 pm) nd retl temperture ( C) throughout the proedure. Two dimensionl, M-mode, nd spetrl Doppler Imging ws performed using the VisulSonis Vevo 77 ultrsound system with the RMV 77 trnsduer (VisulSonis, Toronto, Ontrio, Cnd). At the ompletion of the snning, the mouse ws removed from nesthesi nd llowed to wke up prior to returning to the olony. Grip strength test. Grip strength ws ssessed using grip strength meter onsisting of horizontl forelim mesh nd n ngled hind lim mesh (Columus Instruments, Columus OH). Five suessful hindlim nd forelim strength mesurements within 2 minutes were reorded, nd dt ws normlized to ody weight nd expressed s kilogrm fore. Mesurement of serum CK nd other omponents. Mouse lood ws tken immeditely fter ervil dislotion nd entrifuged t 1,5 r.p.m. for 3 minutes. Serum ws seprted nd stored t 8 C. The level of serum omponents ws determined y Chrles Riverside Lortories (Columi, MO). In vivo rdi hemodynmis. Hemodynmis nlysis on the treted nd untreted mdx s well s the C57BL/6 mie were performed using ondutne miromnometry ording to previously pulished methods Briefly, the mie were nesthetized y inhltion of 1% isoflourne nd 99% oxygen. The mie were then ventilted t 12 strokes/ minute with rodent ventiltor (Hrvrd Instruments, Holliston, MA) nd kept on wrm pt to stilize the ody temperture t 37 C. A 1.4 F high-fidelity miromnometer theter (Millr Instruments, Houston, TX) ws inserted into the left ventrile through rotid rtery under ontinuous hemodynmi monitoring. Following olletion of seline hemodynmi dt, doutmine ws infused t rte of 6.5 µg/g/min for 3 minutes through the jugulr vein. Pressure-volume loops dt were olleted online t 5 Hz. Hemodynmi prmeters were nlyzed with PVAN3.3 softwre (Millr Instruments). SUPPLEMENRY MATERIAL Figure S1. Exmintion of ollgen deposition in musles y pirosirius stining. ACKNOWLEDGMENTS This work ws supported y the Crolins Musulr Dystrophy Reserh Endowment t the Crolins HelthCre Foundtion nd Crolins Medil Center (Chrlotte, NC); US Army Medil Reserh, Deprtment of Defense (W81XWH ). The uthors delred no onflit of interest. REFERENCES 1. Hoffmn, EP, Brown, RH Jr nd Kunkel, LM (1987). Dystrophin: the protein produt of the Duhenne musulr dystrophy lous. Cell 51: Ling, NG (1993). Moleulr nd Cell Biology of Musulr Dystrophy. In: Prtridge, (ed.). Chpmn: London, pp Mono, AP, Bertelson, CJ, Liehti-Gllti, S, Moser, H nd Kunkel, LM (1988). An explntion for the phenotypi differenes etween ptients ering prtil deletions of the DMD lous. Genomis 2: Athnsopoulos, T, Grhm, IR, Foster, H nd Dikson, G (24). Reominnt denossoited virl (raav) vetors s therpeuti tools for Duhenne musulr dystrophy (DMD). 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Prevention of dystrophin-defiient rdiomyopthy in twenty-one-month-old rrier mie y mosi dystrophin expression or omplementry dystrophin/utrophin expression. Cir Res 12: Burger, AJ, Notrinni, MP nd Aronson, D (2). Sfety nd effiy of n elerted doutmine stress ehordiogrphy protool in the evlution of oronry rtery disese. Am J Crdiol 86: Townsend, D, Blnkinship, MJ, Allen, JM, Gregorevi, P, Chmerlin, JS nd Metzger, JM (27). Systemi dministrtion of miro-dystrophin restores rdi geometry nd prevents doutmine-indued rdi pump filure. Mol Ther 15: Ysud, S, Townsend, D, Mihele, DE, Fvre, EG, Dy, SM nd Metzger, JM (25). Dystrophi hert filure loked y memrne selnt poloxmer. Nture 436: Moleulr Therpy vol. 19 no. 3 mr