Systemic delivery of genes to striated muscles using adeno-associated viral vectors

Transcription

1 24 Nture Pulishing Group Systemi elivery of genes to strite musles using eno-ssoite virl vetors Pul Gregorevi,4,Mihel J Blnkinship,4,Jmes M Allen 2,Roert W Crwfor,Leonr Meuse, Dniel G Miller 2,Dvi W Russell 2,3 & Jeffrey S Chmerlin 3 A mjor ostle limiting gene therpy for iseses of the hert n skeletl musles is n inility to eliver genes systemilly to musles of n ult orgnism Systemi gene trnsfer to strite musles is hmpere y the vsulr enothelium, whih represents rrier to istriution of vetors vi the irultion Here we show the first eviene of wiespre trnsution of oth ri n skeletl musles in n ult mmml, fter single intrvenous ministrtion of reominnt enossoite virus pseuotype 6 vetors The inlusion of vsulr enothelium growth ftor/vsulr permeility ftor, to hieve ute permeiliztion of the peripherl mirovsulture, enhne tissue trnsution t lower vetor oses This tehnique enle wiespre musle-speifi expression of funtionl miro-ystrophin in the skeletl musles of ystrophinefiient mx mie, whih moel Duhenne musulr ystrophy We propose tht these methos my e pplile for systemi elivery of wie vriety of genes to the strite musles of ult mmmls Humn mortlity n qulity of life re signifintly ffete y iseses of the strite musulture Geneti tretments tht re eing evelope for onitions suh s hert isese, ging-ssoite musle wsting n the musulr ystrophies hve een limite y n inility to hieve wiespre n effiient gene trnsfer to the hert n isperse skeletl musles of n ult orgnism 4 For exmple, nesthesi, invsive surgery n hzrous oftors re require to trnsue vrying frtions of the riomyoyte popultion effiiently,5 Similrly, the trnsfer of genes to the musles of iniviul lims using vrious vetors requires either iret injetion of iniviul musles, or omplex surgil proeures performe uner nesthesi to istriute vetors vi the irultion 2 4,6 Here we esrie simple n highly effiient metho to trnsfer genes systemilly to the ri n skeletl musles of ult mmmls This pproh uses intrvenous ministrtion of reominnt eno-ssoite virus pseuotype 6 (raav6-pseuotype vetors),whih re extremely effetive t trnsuing skeletl musles fter intrmusulr injetion 2 RESULTS Systemi trnsution of skeletl musles y raav6 First, we exmine the potentil for systemi gene trnsfer fter intrvenous ministrtion of raav6 vetors t the whole-oy level in young ult (6 8 wk) C57Bl/J mie (Fig ) The musles of mie exmine ys fter ministrtion through the til vein of 2 vetor genomes of raav6 vetor ontining CMV lz expression ssette, i not show ovious exogenous β-gltosise (β-gl) tivity ompre with the strite musles of untrete mie, emonstrting the iffiulties in hieving systemi gene trnsfer (Fig ) Beuse vsulr permeilizing gents hve een shown to influene extrvsulr issemintion of vrious vetors, we surveye previously hrterize,4,5,8 ompouns for enhnement of trnsution effiieny Of the gents exmine (inluing histmineppverine omintions n oministrtion of enosine), systemi oministrtion of vetor n vsulr enothelil growth ftor (VEGF), potent effetor of mirovsulr permeility 3,4,proue the gretest enhnement of reporter gene expression Mie ministere 2 vetor genomes of raav6 CMV lz vetor omine with µg of VEGF emonstrte extensive β-gl expression simultneously in numerous xil n ppeniulr musles (Fig ) Mie ministere higher vetor ose ( 2 vetor genomes) showe even greter trnsution of skeletl musles, hieving extensive expression of β-gl throughout the entire skeletl musulture (Fig ) These t emonstrte for the first time metho for hieving gene trnsfer throughout the skeletl musles of n ult niml through single intrvsulr ministrtion of reominnt vetor An immunologil retion n sometimes e oserve fter foreign gene trnsfer to skeletl musles, whih is the result of expression of the trnsgene in non-musle ntigen-presenting ells 5,6 The esign of expression ssettes inorporting trnsription-regulting element tht onstrins trnsgene expression to skeletl musle ells represents metho of minimizing trnsgene expression in non-musle ells The engineere regultory element CK6 is one exmple of musle-speifi trnsription ontrol element tht n e inorporte into expression ssettes intene for elivery y raav vetors 5 However, this promoter is only % s tive s the CMV promoter in skeletl musles in vivo 5 To ssess the reporter gene tivity Deprtments of Neurology, 2 Meiine n 3 Biohemistry, Sentor Pul D Wellstone Musulr Dystrophy Coopertive Reserh Center, The University of Wshington, Settle, Wshington 9895, USA 4 These uthors ontriute eqully to this work Corresponene shoul e resse to JSC (js5@uwshingtoneu) Pulishe online 25 July 24; oi:38/nm VOLUME NUMBER 8 AUGUST 24 NATURE MEDICINE

2 Figure Musle trnsution (lue) following systemi raav6 ministrtion ( e) Mie were exmine t ( ) or 4 (,e) ys fter ministrtion of 2 (,), 2 (, ) or 3 (e) vetor genomes of raav6 CMV lz ( ) or raav6 CK6 lz (,e) lone () or with µg VEGF ( e) 24 Nture Pulishing Group e regulte y the CK6 musle-speifi expression element in the skeletl musles of ult mie, we ministere either 2,or 3 vetor genomes of raav6 CK6 lz vetors to mie through the til vein At 4 ys post-tretment, inrese β-gl expression ws oserve in xil n ppeniulr musles ross the oies of mie trete with 2 vetor genomes of raav6 CK6 lz (Fig ) However, the pttern of expression otine using raav6 CK6 lz ws reue ompre with mie ministere n equivlent ose of raav6 CMV lz In ft, it ws neessry to minister 3 vetor genomes of raav6 CK6 lz n VEGF to mie to hieve oy-wie trnsution omprle to tht oserve in mie ministere one-tenth the ose of raav6 CMV lz n VEGF (Fig e) To ssess quntittively the trnsution of musles in mie ministere raav6 vetors through intrvenous injetion, iniviul musles of mie from eh ohort were homogenize n ssye for β-gl tivity Consistent with the erlier oservtions, the β-gl tivity of the musles of mie exmine ys fter ministrtion of 2 vetor genomes of the raav6 CMV lz vetor i not iffer from tht of the musles of untrete mie (Fig 2) However, mie ministere 2 vetor genomes of raav6 CMV lz vetor long with µg VEGF emonstrte fol inrese in β-gl tivity in ri n vrious skeletl musles ompre with mie reeiving only the vetor (Fig 2) Mie ministere 2 vetor genomes of raav6 CMV lz emonstrte further inreses in trnsution levels: etween 8, fol greter in ri n vrious skeletl musles thn in untrete mie (Fig 2) Similr trnsution effiienies were hieve with vetor oses of 2 vetor genomes in the presene n sene of VEGF, emonstrting tht the mehnism of inrese trnsution effiieny with VEGF oministrtion is less influentil over the rnge of VEGF oses teste, s the vetor ose is inrese The β-gl tivity mesure in the ppeniulr skeletl musles of mie ministere 3 vetor genomes of raav6 CK6 lz n VEGF ompre fvorly with levels oserve in mie ministere 2 vetor genomes of raav6 CMV lz with VEGF (Fig 2) Notly, nimls ministere raav6 CK6 lz vetors showe little β-gl tivity in the hert n iphrgm musles, regrless of vetor ose 5, lthough signifint trnsgene expression in these tissues is lerly hievle when vetors with CMV promoter re use (Figs n 2) As single intrvenous ministrtion of raav6 vetors hieve mrke trnsgene expression in skeletl musles throughout the oy of n ult niml (Fig 2,), we hose to histologilly exmine iniviul musles to etermine the extent of trnsution t the ellulr level Immunofluoresene mirosopy highlighte wiespre trnsgene expression throughout the musle fiers omprising the musles of mie ministere 2 vetor genomes of raav6 CMV lz plus VEGF (Fig 2) Intrvenous ministrtion of raav6 vetors therefore hieves not only oy-wie issemintion of vetor, ut lso reltively uniform gene trnsfer throughout the mjority of the skeletl musle fier popultion This is espeilly notle when onsiering tht the issemintion of vetors ministere y mens of iret intrmusulr elivery n e hmpere y musle ntomy n extrellulr omponents suh s onnetive tissue Correltion etween trnsution n vetor genomes In orer to further investigte the effets of VEGF on the trnsution of strite musles of n ult niml fter intrvenous ministrtion of raav6 vetors, we exmine β-gl tivity n vetor genome opy numers in ri n skeletl musle smples otine from mie oministere ifferent oses of VEGF t the time of vetor ministrtion The β-gl tivity of ri n skeletl musles from mie ministere 2 vetor genomes of raav6 CMV lz inrese ose-epenently with inresing quntities of oministere VEGF (Fig 3) Quntifition of vetor genome opy numers in hert n tiilis nterior musle smples emonstrte onsistent tren etween reporter gene tivity n vetor genome opies over the rnge of VEGF oses exmine (Fig 3,) These t emonstrte tht oministrtion of VEGF with raav6 vetors, ministere y mens of single intrvenous injetion, inreses the umultion of vetor genome opies in iniviul musles In ontrst to the hert n skeletl musles, elevte β-gl tivity ws not oserve in rin, lung, liver, spleen, intestine, kiney or testes smples hrveste from trete mie (Fig 3) However, raav6 vetor genomes were etete in homogentes of these tissues fter intrvenous ministrtion of vetor with VEGF (Fig 3), whih is onsistent with previous stuies 7 Furthermore, heptoytes n e trnsue y intrvenously-ministere raav6 vetors ontining liver-speifi gene regultory elements 7 Therefore, intrvenous ministrtion of raav6 NATURE MEDICINE VOLUME NUMBER 8 AUGUST

3 24 Nture Pulishing Group RLU(mg prot ) RLU(mg prot ),,,, M FL Bi Tr Ht Di Q TA G So M FL Bi Tr Ht Di Q TA G So vetors my prove useful for trnsuing numerous tissue types, epening on the esign of the expression ssette Importntly, serum-se inies of orgn funtion were omprle etween ohorts reeiving vehile, vehile with VEGF, empty psis with VEGF n vetor with VEGF, emonstrting tht the intervention regimen i not inue ute orgn toxiity (Supplementry Fig online) raav6 vetors potently trnsue the myorium in vivo In ition to showing uniform lz expression in the vst mjority of skeletl musle fiers throughout the oy (Fig 2), mie reeiving RLU(mg prot ),, raav6 CMV lz with VEGF lso emonstrte rmti trnsution of the riomyoyte popultion (Fig 4) The trnsution levels oserve in the hert exeee those reporte for the most promising of the existing methos,4 6, wheres skeletl musle trnsution ws equl to, or exeee, the est results otine previously using pprohes tht trget iniviul musles in isolte lims 2 4,6,2 As the hert hs een regre s n extremely iffiult orgn in whih to hieve wiespre geneti trnsfer,5, the extensive n uniform trnsution oserve in the myorium lone is prtiulrly enourging Furthermore, lterntive methos of gene trnsfer hve not een le to simultneously trget the wiely isperse xil n ppeniulr strite musles in n ult mmml Although rmti trnsution of the myorium ws hieve in mie ministere raav6 CMV lz (Fig 4), ompre with mie ministere vehile (Fig 4,) or n equivlent ose of raav6 vetor psi lking n expression ssette (empty psis; Fig 4,e), histologil inspetion revele signifint mononuler ell infiltrtes in the myorium of some wil-type mie pproximtely 4 ys fter ministrtion of the raav6 CMV lz vetor (Fig 4f) Cellulr infiltrtion ws ssoite with the umultion of CD4-positive ells, whih typilly inlue T-helper lymphoytes (Fig 4g) As ministrtion of n equivlent ose of raav6 empty psis with VEGF i not inue mononuler ell infiltrtes (Fig 4,e), we hypothesize tht the inflmmtory retion oserve with CMV lz in wil-type mie results from the onstitutive wiespre expression of teril protein (β-gl) 8 Inee, ministrtion of 2 vetor genomes of raav6 CMV lz n VEGF to trnsgeni mie tht express teril β-gl within the intestinl villi 9, i not use mononuler ell umultion (Fig 4h,i), n mie trete M FL Bi Tr Ht Di Q TA G So Figure 2 β-gl tivity in musles fter systemi raav6 ministrtion Brs represent tivity (RLU: reltive light units) from mie ministere: () 2 vetor genomes raav6 CMV lz (gry, only soleus ove kgroun P 5 with Stuent s t-test) or vetor with µg of VEGF (re); () 2 vetor genomes of raav6 CMV lz (lk) or vetor n µg VEGF (rk re); () 2 vetor genomes (light lue) or 3 vetor genomes (rk lue) of raav6 CK6 lz, oth with µg VEGF Dt from uninjete nimls (white rs) re shown in Dt represent mens ± se M, msseter; FL, flexor igitorum profunus/rpi rilis; Bi, ieps; Tr, trieps; Ht, hert; Di, iphrgm; Q, qurieps; TA, tiilis nterior; G, gstronemius; So, soleus (), Anti-β-gl immunofluoresene mirosopy of the qurieps musles : mouse ministere 2 vetor genomes of raav6 CMV lz y intrvenous injetion ( n inset right), ompre with n uninjete mouse (, inset left) Animls exmine (,,) or 4 () ys post-tretment Brs, mm () n µm Figure 3 β-gl tivity n vetor genome opy numers in musles n orgns fter systemi ministrtion of raav6 vetors with VEGF (,) β-gl tivity () n raav genome opy numer () in strite musles fter intrvenous injetion of 2 vetor genomes of raav6 CMV lz oministere with (yellow; genomes not etermine for TA), 5 (ornge), or (re) µg of VEGF, ompre with no VEGF ministrtion (gry); ritrry unit = 8 4 vetor genomes µg/dna () β-gl tivity in vriety of orgns fter intrvenous injetion of 2 vetor genomes of raav6 CMV lz lone (lk) or with µg of VEGF (rk re) () raav6 genomes re etetle in orgns from ; ritrry unit = 9 6 vetor genomes/µg DNA Dt from uninjete nimls (white rs) re shown in n Dt re men vlues ± se TA, tiilis nterior; Br, rin; Lu, lung; Li, liver; Sp, spleen; In, intestine; Ki, kiney; Te, testes Animls were exmine ys post-tretment RLU (mg prot) Reltive vetor opies (µg DNA),, Hert Hert TA TA RLU (mg prot) Reltive vetor opies (µg DNA),, Br Lu Li Sp In Ki Te Br Lu Li Sp In Ki Te 83 VOLUME NUMBER 8 AUGUST 24 NATURE MEDICINE

4 ARTICLES 24 Nture Pulishing Group e f g h i Figure 4 Myoril trnsution (lue) in ult mie ys fter systemi ministrtion of 2 vetor genomes of raav6 CMV lz n µg VEGF () The hert of trete wil-type mouse exmine fter retion with X-gl n ounterstining with hemtoxylin n eosin ( i) Setions exmine following retion with X-gl n ounterstining with hemtoxylin n eosin (,,f,h) n seril setions immunofluoresene-lele for CD4-positive ells (re;,e,g,i) The herts of n untrete mouse (,) n mouse ministere empty vetor psis with VEGF (,e) isply regulr morphology n few CD4postive ells (f,g) Regions of myorium in the hert of wil-type mouse ministere 2 vetor genomes of raav6 CMV lz n VEGF show rmti mononuler ell n CD4-postive ell infiltrtion (h,i) Myorium of trete, β-gl-tolernt trnsgeni mouse (villin mouse9), emonstrting lk of ellulr infiltrte Sle rs, mm () n µm ( i) with the raav6 CK6 lz vetor i not show signifint mononuler ell umultion or histologil normlities in strite musles (t not shown) These t estlish tht the mononuler ell infiltrte oserve in wil-type mie is not ssoite with virl omponents of the raav6 vetor, n tht high oses of raav6 vetors expressing β-gl from musle-speifi promoter re tolerte well y immunoompetent ult mmmls Resue of isese moel y systemi raav6 elivery To ssess the potentil for hieving systemi trnsution of the skeletl musulture with therpeuti trnsgene, we stuie the ystrophin-efiient mx mouse moel of Duhenne musulr ystrophy2 Ptients with Duhenne musulr ystrophy ie premturely from profoun ri n respirtory musle egenertion, owing to n inility to proue ystrophin, whih stilizes the musle fier rhiteture2 Strtegi engineering of the ystrophin oing sequene hs rete trunte, yet highly funtionl, miroystrophin DNA ssettes tht n e rrie y raav vetors3, To etermine whether raav6 oul eliver these funtionl miroystrophins in systemi mnner, mx mie were injete with 2 vetor genomes of raav6 CK6 miroystrophin ( R4 23/ CT)3 n VEGF (Fig 5) Eight weeks fter tretment, wiespre ystrophin expression ws oserve throughout the skeletl musles of mie (Fig 5) with the exeption of the iphrgm, where the CK6 promoter ws not tive (t not shown)5 A hrteristi feture of musles of the mx mouse is n inrese suseptiility to ontrtion-inue injury9,2 We n others hve shown tht expression of highly funtionl, miroystrophin proteins in the musles of mx mie n hlt the progression of the pthology n improve the ystrophi phenotype3,,22 The NATURE MEDICINE VOLUME NUMBER 8 AUGUST 24 tiilis nterior lim musles of trete mie emonstrte reue suseptiility to injury y protool of eentri ontrtions9 ompre with untrete mx mie (Fig 5), whih ompres fvorly with t otine following optimize, iret injetion of the tiilis nterior musle with vetors expressing trunte or full-length ystrophins9, Despite the signifintly improve funtionl performne of the tiilis nterior musles of trete mx mie, phenotypi orretion ws inomplete Some spets of musle funtion suh s solute (Po) n normlize (spo) fore-generting pity i not iffer etween the musles of trete n untrete mx mie (men ± stnr error (se): wil-type, Po 2 ± 5 mn, spo 246 ± 5 kn m 2; mx, Po,234 ± 65 mn, spo 82 ± 8 kn m 2; trete mx Po,3 ± 5 mn, spo 87 ± kn m 2) Although ner omplete phenotypi orretion is oserve in trnsgeni mie expressing miroystrophin3, mximl orretion of the ystrophi phenotype my only our fter progressive n extene remoeling of the skeletl musulture in the trete niml, n some spets of the pthologil phenotype tht evelop erly in life my not e fully reversile This hypothesis hs een supporte in stuies emonstrting tht trnsgeni mie expressing full-length ystrophin only fter irth hlt isese progression ut fil to hieve the mgnitue of phenotypi orretion oserve in mie expressing ystrophin uring emryogenesis23, result similr to the present finings Prtil mosi expression oserve in the tiilis nterior musle t this vetor ose (Fig 5) my lso hve ontriute to the inomplete phenotypi orretion However, previous stuies of mx mie hve emonstrte tht ystrophin expression t levels greter thn 2% of wil-type expression in mjority of musle fiers, level gretly exeee in these stuies, profounly improves the isese phenotype3,24 83

5 ARTICLES Forelim Wil type Wil type Interostl Quriep,2 i 8 6 ii mx 4 2 Tmx % of initil Po 24 Nture Pulishing Group mx Po(mN), 8 iii 6 iv 4 Tmx 2 LC LC2 Figure 5 Systemi elivery of miroystrophin to ystrophi mie () Anti-ystrophin immunofluoresene mirosopy of tiilis nterior musles from trete mx mie (Tmx) ministere 2 vetor genomes of raav6 CK6 miroystrophin n µg VEGF, ompre with wil-type n untrete ontrols Dystrophin expression is inrese in the musles of trete ompre with untrete mx mie, ut remins mosi ompre with wil-type mie () Fore prouing pity (Po, top) n efiit (ottom), of tiilis nterior musles from Tmx mie (re) ompre with wil-type (lk) n untrete mx (gry) mie fter onseutive eentri ontrtions (LC, LC2, respetively)9 P vlues with Stuent s t-test etween Tmx n mx: i = 78, ii 5, iii = 74 n iv 5 () Anti-ystrophin-lele musles from mie ministere 3 vetor genomes of raav6 CK6 miroystrophin n VEGF The ystrophin expression pttern oserve t this vetor ose is no longer mosi, in ontrst to Mie were exmine t 8 weeks (,) n 6 weeks () fter tretment Sle rs, µm Boy-wie expression of raav6-elivere ystrophin To etermine whether uniform ystrophin expression oul e otine fter intrvsulr vetor elivery, n itionl ohort of mx mie were injete with tenfol greter vetor ose ( 3 vetor genomes) n VEGF Six weeks post-tretment, uniform expression of the trunte ystrophin protein ws oserve throughout musles of the lims n torso (inluing the interostl musles) of mie, t levels similr to tht of the norml protein in wil-type mie (Figs 5 n 6) This is the first stuy to emonstrte expression of therpeuti ystrophin-se proteins simultneously in xil n ppeniulr skeletl musulture ross the oy of n ult ystrophi niml fter vetor ministrtion Trete ystrophi mie lso isplye n 5% reution in serum retine kinse levels (P < 5) ompre with untrete ystrophi mie, emonstrting oy-wie phenotypi hnge initive of reue musle egenertion fter tretment (Fig 6) Despite hieving wiespre trnsfer of n expression ssette inorporting trnsgene of humn origin, there ws no histopthologil eviene of mononuler ell umultion in these musles, whih express the therpeuti trunte ystrophin protein uner the ontrol of musle-speifi trnsriptionl regultor (Fig 6) These t emonstrte tht systemi ministrtion of raav6 vetors inorporting musle-speifi expression ssette n hieve sustine expression of therpeuti protein throughout the skeletl musles of n niml moel of musulr ystrophy DISCUSSION Ahieving effiient issemintion of vetors throughout the musles of the oy hs proven mjor hllenge to ttempts to genetilly 832 mnipulte strite musle ells The finings of the present stuy emonstrte for the first time tht extensive trnsution of the myorium n skeletl musulture of n ult mmml is hievle y mens of single intrvenous ministrtion of raav6 vetors Consistent with previous reports7, the trnsution effet seems to e influene y vetor ose, n to e sujet to threshol event, s some vetor oses show signifint expression in the hert n skeletl musle fiers only when oministere with vsulturepermeilizing gent, in this se VEGF (Figs n 2) Wiespre trnsution of strite musulture ws hieve without ute toxiologil response using oses of vetor (etween 2 n 2 vetor genomes) similr to intrvenous n intrmusulr oses of raav2 tolerte y lrger nimls25,26 Also, ptients hve een ministere intrmusulr injetions of greter thn 4 raav2 vetor genomes with no verse effets27 VEGF oministrtion ws tolerte well y ll mie, inluing t oses up to twofol higher thn presente here (Supplementry Fig online; t not shown) The effetive VEGF ose rnge is out the sme s oses ministere to lrger nimls, whih re within n orer of mgnitue of tretments suessfully ministere to ptients28 As systemi lerne of VEGF is rpi n ute, VEGF-inue vsulr hnges re lrgely shortlive28; VEGF oministrtion is espeilly enourging s mens to enhne systemi gene elivery fter intrvsulr ministrtion of vetors to onsious mmmls using reue vetor ose Aministrtion of raav6 vetors rrying lz or miroystrophin regulte y the musle-speifi CK6 promoter/enhner5 ws tolerte reily y mie for the urtion of this stuy (t lest 8 weeks), highlighting the importne of mintining tissue-speifi gene VOLUME NUMBER 8 AUGUST 24 NATURE MEDICINE

6 ARTICLES Figure 6 Sustine expression of therpeuti humn miroystrophin protein is hieve fter intrvenous ministrtion of raav6 vetors, without signifint ellulr infiltrtion () Anti-ystrophin-lele tiilis nterior from trete mx mouse ministere 3 vetor genomes raav6 CK6 miroystrophin n µg VEGF This tretment signifintly (Tmx ompre with mx, P 5) reue serum retine kinse (CK) levels Iniviul t re epite s ots with group mens in re () Anti-ystrophin immunofluoresene mirosopy (left) n hemtoxylinstine n eosin-stine setions (right) of tiilis nterior musles from wil-type, untrete mx (mx) n mx mie trete s in (Tmx), respetively Yellow points funtion re mrkers ientifying the sme musle fier in left n right pnels Mie were exmine 6 weeks fter tretment Sle rs, mm () n 2 µm () 4, 2,, WT mx 2Tmx 3 Wil type mx Tmx 24 Nture Pulishing Group 3, expression We hve shown tht systemi ministrtion of raav6 vetors ontining therpeuti trnsgene n lso hieve wiespre n sustine expression of struturl protein in the vst mjority of the skeletl musulture in murine moel of musulr ystrophy (Figs 5 n 6) This level is suffiient to onfer improvement of the isese phenotype (Figs 5 n 6) Tretment of most musle iseses will most proly require sustine gene expression raav-meite gene expression hs een emonstrte for the life of trete mie, n t the longest time points stuie presently in nine sujets (eyon 4 yers)27,29 Although this pproh hs n ovious potentil pplition to the vrious forms of musulr ystrophy, there oul lso e potentil pplitions where geneti trnsfer to the myorium n/or skeletl musle fiers is wrrnte for the tretment of other humn ptholo- NATURE MEDICINE VOLUME NUMBER 8 AUGUST 24 gies, or for generting experimentl niml moels of gene expression Potentil linil pplitions inlue trnsution of the musulture to express seretle or trophi ftors, suh s for the tretment of ri pthologies, hemophili7,26,27 or musle wsting ssoite with ging2 METHODS Construt loning n vetor proution Reominnt AAV genomes ontining expression ssettes for β-gl or miroystrophin ( R4 23/ CT)3 n serotype 2 inverte terminl repets were generte y stnr loning tehniques3 EgI frgments of the oing sequenes were lone into the EgI site of plsmis pdd2 (ontining the CMV promoter) for β-gl n pdd344 (ontining the CK6 promoter5) for oth oing sequenes3 The pkging/helper plsmi pdgm6, whih inlues the serotype 6 psi reing frme, ws generte y mplifying the psi gene from lone of AAV serotype 6 (ref 3) y PCR using the oligonuleotie primers 5 - ATTTAAATCAGGTATGGCTGCCGATGGT-3 n 5 -ATCGATTGCTATGGTGACCAGATAAGATAA-3 enoing terminl SwI n ClI restrition sites n inserting the resulting DNA frgment into plsmi pdg (ref 3) ut with the sme enzymes HEK293 ells were plte t ensity of ells on -m ulture ish, 8 6 h prior to trnsfetion with µg of vetorgenome-ontining plsmi n 2 µg of the pkging/helper plsmi pdgm6, y mens of the lium phosphte preipitte metho to generte pseuotype 6 vetors,3 Seventy-two hours fter trnsfetion, the mei n ells were ollete n homogenize through mirofluiizer (Mirofluiis) prior to 22-µm lrifition (Millipore) The vetor ws purifie from the lrifie lyste y ffinity hromtogrphy32 over HiTrp heprin olumn (Amershm), n ilyze to physiologil Ringer s solution The purifie virus ws titere with quntittive hemiluminesent slot-lot ginst the vetor genome, visulize with the CDP-Str kit (Amershm) n GeneGnome imge quisition/nlysis system (Syngene Bio Imging) Mnipultion of experimentl nimls All experimentl mnipultion of young ult (6 8 week) mle wil-type C57Bl/J, or ystrophi C57Bl/SSn Dmmx/J mie (Jkson Ls) ws pprove y the Institutionl Animl Cre n Use Committee of the University of Wshington Iniviul mie (from ohorts ontining 4 6 nimls) were ministere 3 µl olus injetion (orresponing to 5% of the irulting loo volume, reily tolerle ose) through the til vein omprising physiologil Ringer s solution ontining up to 3 vetor genomes ( 4 4 vetor genomes/kg) of raav6 vetor, 2 µg ( 8 µg/kg) reominnt humn VEGF-65 (R&D Systems), 8% mouse serum lumin (Sigm) n 2 IU (interntionl units) (8 IU/kg) soium heprin Tissue proessing n nlysis For whole-mount retion of lim n torso segments for β-gl tivity, tissues were fixe in 4% formlehye-supplemente PBS for 5 min efore inution t 37 C for 8 h in PBS supplemente with 5-romo-4-hloro-3-inolyl β-d-gltopyrnosie (X-gl; Sigm), mgnesium hlorie, n Fe(II,III)CN (potssium ferro/ferriynie) After ompletion of retion inution, tissues were fixe in Krnovsky s solution t 4 C Histologil, histohemil n immunohemil proessing of musle ryosetions were omplete s esrie previously9,33 Immunofluoresene 833

7 24 Nture Pulishing Group mirosopy use primry ntioies ginst teril β-gl (Chemion), CD4 + ells (Phrmingen) n the N-terminl region of ystrophin 33 n the ommeril Alex 488-lele got-nti-rit n Alex 594-lele got-nti-rt seonry ntioies (Moleulr Proes) Protein extrts from the tissues of trete nimls were ssye for β-gl tivity using luminometri kit (BD Biosiene) n mesure for protein onentrtion with the Brfor regent (Piere) Serum retine kinse levels were ssye with kineti NADH formtion kit (Dignosti Chemils Limite) n serum-se hemil inies of hepti n renl funtion were nlyze ommerilly (Phoenix Centrl Lortory) Reltive numers of vetor genomes were etermine using rel-time PCR on n ABI Prism 77 (Perkin Elmer) with the following primer-proe omintion (Applie Biosystems) trgeting the lz open reing frme: forwr, 5 -GCGTTACCCAACTTAATCGCC-3 ; reverse, 5 -GCCTCTTCGCTATTACGCCA-3 ; proe, 5 -FAM-CAGCACATCCC- CTTTCG CCA-TAMRA-3 Totl DNA ws quntifie with Hoehst ye fluoresene (BioR) The raav genomes re reporte s reltive numers per µg of DNA minus kgroun, with the highest opy numer smple in eh experiment eing ritrrily ssigne vlue of Funtionl nlysis Eight weeks post-injetion, mx mie trete with 2 vetor genomes of raav6 CK6 miroystrophin ( R4 23/ CT; together with untrete mx n wil-type ontrol mie) were nesthetize with 2,2,2-triromoethnol (Sigm) n ssye in situ for fore genertion n protetion from ontrtion-inue injury s previously esrie 9 After funtionl testing, nimls were srifie n tissues were rpily exise n proesse for histology s ove Note: Supplementry informtion is ville on the Nture Meiine wesite ACKNOWLEDGEMENTS We thnk SD Hushk for helpful isussions; D Dun for the AAV MCS vetor plsmis pdd2 n pdd344; J Kleinshmit for the pkging plsmi pdg; AD Miller for the pkging ell line 293; J Hn for the raav6 CK6 lz onstrut; n M Hrguhi n S Okley for tehnil ssistne This work ws supporte y grnts from the Ntionl Institutes of Helth n the Musulr Dystrophy Assoition (USA) to JSC COMPETING INTERESTS STATEMENT The uthors elre ompeting finnil interests (see the Nture Meiine wesite for etils) Reeive 23 Ferury; epte 29 June 24 Pulishe online t Hoshijim, M et l Chroni suppression of hert-filure progression y pseuophosphorylte mutnt of phospholmn vi in vivo ri raav gene elivery Nt Me 8, (22) 2 Musro, A et l Lolize Igf- trnsgene expression sustins hypertrophy n regenertion in senesent skeletl musle Nt Genet 27, 95 2 (2) 3 Hrper, SQ et l Moulr flexiility of ystrophin: implitions for gene therpy of Duhenne musulr ystrophy Nt Me 8, (22) 4 Greelish, JP et l Stle restortion of the sroglyn omplex in ystrophi musle perfuse with histmine n reominnt eno-ssoite virl vetor Nt Me 5, (999) 5 Briges, CR et l Glol ri-speifi trnsgene expression using riopulmonry ypss with ri isoltion Ann Thor Surg 73, (22) 6 Hur, J et l The route of ministrtion is mjor eterminnt of the trnsution effiieny of rt tissues y enovirl reominnts Gene Ther 2, 7 5 (995) 7 Cho, WK et l Moultion of Strling fores n musle fier mturity permits enovirus-meite gene trnsfer to ult ystrophi (mx) mie y the intrvsulr route Hum Gene Ther, 7 74 (2) 8 Zhng, G, Buker, V, Willims, P, Hnson, K & Wolff, JA Surgil proeures for intrvsulr elivery of plsmi DNA to orgns Methos Enzymol 346, (22) 9 Dellorusso, C et l Funtionl orretion of ult mx mouse musle using gutte enovirl vetors expressing full-length ystrophin Pro Ntl A Si USA 99, (22) Wthko, J et l Aeno-ssoite virus vetor-meite miniystrophin gene therpy improves ystrophi musle ontrtile funtion in mx mie Hum Gene Ther 3, (22) Hlert, CL, Rutlege, EA, Allen, JM, Russell, DW & Miller, AD Repet trnsution in the mouse lung y using eno-ssoite virus vetors with ifferent serotypes J Virol 74, (2) 2 Sott, JM et l Virl vetors for gene trnsfer of miro-, mini-, or full-length ystrophin Neuromusul Disor 2, S23 S29 (22) 3 Roerts, WG & Ple, GE Inrese mirovsulr permeility n enothelil fenestrtion inue y vsulr enothelil growth ftor J Cell Si 8, (995) 4 Senger, DR, Perruzzi, CA, Feer, J & Dvork, HF A highly onserve vsulr permeility ftor serete y vriety of humn n roent tumor ell lines Cner Res 46, (986) 5 Huser, MA et l Anlysis of musle retine kinse regultory elements in reominnt enovirl vetors Mol Ther 2, 6 25 (2) 6 Hrtign-O Connor, D, Kirk, CJ, Crwfor, R, Mule, JJ & Chmerlin, JS Immune evsion y musle-speifi gene expression in ystrophi musle Mol Ther 4, (2) 7 Grimm, D et l Prelinil in vivo evlution of pseuotype eno-ssoite virus vetors for liver gene therpy Bloo 2, (23) 8 Yus, K et l Aeno-ssoite virus vetor-meite gene trnsfer into ystrophinefiient skeletl musles evokes enhne immune response ginst the trnsgene prout Gene Ther 9, (22) 9 Pinson, KI, Dunr, L, Smuelson, L & Gumuio, DL Trgete isruption of the mouse villin gene oes not impir the morphogenesis of mirovilli Dev Dyn 2, 9 2 (998) 2 Petrof, BJ, Shrger, JB, Stemn, HH, Kelly, AM & Sweeney, HL Dystrophin protets the srolemm from stresses evelope uring musle ontrtion Pro Ntl A Si USA 9, (993) 2 Gregorevi, P & Chmerlin, JS Gene therpy for musulr ystrophy - review of promising progress Expert Opin Biol Ther 3, (23) 22 Skmoto, M et l Miro-ystrophin DNA meliortes ystrophi phenotypes when introue into mx mie s trnsgene Biohem Biophys Res Commun 293, (22) 23 Ahm, A, Brinson, M, Hoges, BL, Chmerlin, JS & Amlfitno, A Mx mie inuily expressing ystrophin provie insights into the potentil of gene therpy for Duhenne musulr ystrophy Hum Mol Genet 9, (2) 24 Phelps, SF et l Expression of full-length n trunte ystrophin mini-genes in trnsgeni mx mie Hum Mol Genet 4, (995) 25 Arru, VR et l Lk of germline trnsmission of vetor sequenes following systemi ministrtion of reominnt AAV-2 vetor in mles Mol Ther 4, (2) 26 Herzog, RW et l Influene of vetor ose on ftor IX-speifi T n B ell responses in musle-irete gene therpy Hum Gene Ther 3, (22) 27 Mnno, CS et l AAV-meite ftor IX gene trnsfer to skeletl musle in ptients with severe hemophili B Bloo, (23) 28 Eppler, SM et l A trget-meite moel to esrie the phrmokinetis n hemoynmi effets of reominnt humn vsulr enothelil growth ftor in humns Clin Phrmol Ther 72, 2 32 (22) 29 Herzog, RW et l Long-term orretion of nine hemophili B y gene trnsfer of loo ogultion ftor IX meite y eno-ssoite virl vetor Nt Me 5, (999) 3 Rutlege, EA, Hlert, CL & Russell, DW Infetious lones n vetors erive from eno-ssoite virus (AAV) serotypes other thn AAV type 2 J Virol 72, (998) 3 Grimm, D, Kern, A, Rittner, K & Kleinshmit, JA Novel tools for proution n purifition of reominnt eno-ssoite virus vetors Hum Gene Ther 9, (998) 32 Hlert, CL, Allen, JM & Miller, AD Aeno-ssoite virus type 6 (AAV6) vetors meite effiient trnsution of irwy epithelil ells in mouse lungs ompre to tht of AAV2 vetors J Virol 75, (2) 33 Rfel, JA et l Fore expression of ystrophin eletion onstruts revels struture-funtion orreltions J Cell Biol 34, 93 2 (996) 834 VOLUME NUMBER 8 AUGUST 24 NATURE MEDICINE