Virus-mediated EpoR76E Therapy Slows Optic Nerve Axonopathy in Experimental Glaucoma

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1 The Amerin Soiety of Gene & Cell Therpy originl rtile Virus-meite EpoR76E Therpy Slows Opti Nerve Axonopthy in Experimentl Gluom Wesley S Bon 1,2, Jessi Hines-Ber 1,2, YPul L GolenMerry 1, Mr Dvis 1, Alm Frooque 1, Ree M Sppington 1,2, Dvi J Clkins 1,2 n Toni S Rex 1,2 1 Deprtment of Ophthlmology n Visul Sienes, Vnerilt Eye Institute, Vnerilt University Meil Center, Nshville, Tennessee, USA; 2 Vnerilt Brin Institute, Vnerilt University Meil Center, Nshville, Tennessee, USA Gluom, ommon use of linness, is urrently trete y introulr pressure (IOP) lowering interventions. However, this pproh is insuffiient to ompletely prevent vision loss. Here, we evlute n IOP-inepenent gene therpy strtegy using moifie erythropoietin, EPO-R76E, whih hs reue erythropoieti funtion. We use two moels of gluom, the murine miroe olusion moel n the DBA/2J mouse. Systemi reominnt eno-ssoite virus meite gene elivery of EpoR76E () ws performe onurrent with elevtion of IOP. Axon struture n tive nterogre trnsport were preserve in oth moels. Vision, s etermine y the flsh visul evoke potentil, ws preserve in the DBA/2J. These results show tht systemi EpoR76E gene therpy protets retinl gnglion ells from gluomtous egenertion in two ifferent moels. This suggests tht EPO trgets omponent of the neuroegenertive pthwy tht is ommon to oth moels. The effiy of raav. EpoR76E elivere t onset of IOP elevtion supports linil relevne of this tretment. Reeive 13 July 215; epte 13 Otoer 215; vne online pulition 24 Novemer 215. oi:1.138/mt INTRODUCTION Gluom is the most ommon use of permnent linness worlwie. It is progressive opti neuropthy hrterize y egenertion of retinl gnglion ell (RGC) xons n susequent eth of RGC somt. Although ge is the single most signifint risk ftor for evelopment of gluom, introulr pressure (IOP) is the only moifile risk ftor. Phrmeutil n surgil interventions tht lower IOP slow ut o not lwys hlt progression of the isese. 1,2 In ition, poor ptient ompline in ministering IOP-lowering eye rops is signifint hllenge. Due to the shortomings in IOPlowering therpeuti strtegies, n IOP-inepenent pproh tht loks the neuroegenertion unerlying vision loss in gluom is neee. To voi hllenges of poor ptient ompline with eye rops, new therpy woul ielly hve prolonge therpeuti effet sine gluom is long-term, progressive egenertion. We propose gene therpy, whih n provie long-term, ontinuous elivery of neuroprotetive moleule fter single injetion. Erythropoietin (EPO) is ytokine tht regultes erythropoiesis. After seretion into the systemi irultion y the liver, EPO ins n interts with its ognte reeptor the erythropoietin reeptor (EpoR) homoimer to inhiit poptosis of erythroi progenitor ells n filitte erythroyte proution (for review, see ref. 3). In ition, EPO n its reeptor re proue t lower levels in nonhemtopoieti tissue inluing the retin. 4 In the entrl nervous system, EPO ts on vriety of ell types where it plys role in regulting ell eth, oxitive stress, n neuroinflmmtion (for review, see ref. 5). Due to these pleiotropi effets, EPO possesses signifint therpeuti potentil in neuroegenertive iseses. However, reominnt EPO is of limite utility for the tretment of gluom ue to slow isese progression, the short hlf-life of EPO in vivo, n the inrese in re loo ell proution use y repet tretment with systemi EPO. 6,7 To overome these ostles, we use virl gene elivery of mutnt form of EPO, EPO-R76E, tht hs ttenute erythropoieti tivity. 8,9 Tretment with reominnt eno-ssoite virus (raav) provies sustine, long-term elivery of EPO-R76E without ngerous rise in hemtorit. 8,9 Furthermore, we previously emonstrte tht is neuroprotetive in multiple moels of neuroegenertion. 1,11 We previously showe tht systemi gene elivery of raav. EpoR76E given prior to evelopment of elevte IOP in the DBA/2J mouse moel of pigment ispersion gluom preserve the RGC xons, ell oies, n vision out to 1 months of ge. 7,12 In the urrent stuy, we exten these results in two importnt wys. First, we performe tretment t the onset of IOP inrese, the erliest time point for linil intervention. Seon, we teste therpeuti effiy in n inuile moel of gluom, the murine miroe olusion moel. 13,14 The DBA/2J exhiits neuroinflmmtion in the retin prior to evelopment of elevte IOP, potentilly ue to ltere oulr immune privilege in this strin n oul onfoun gluom stuies. 15 The mouse miroe olusion moel lso moels lose ngle gluom, ut without pigment ispersion or high seline neuroinflmmtion. 13 One of the erliest signs of gluom is efiit in xon trnsport, whih is influene gretly y ge. 13,16 18 In oth moels, xon trnsport efiits preee xon egenertion, whih preees RGC eth. 18 In the DBA/2J in prtiulr, 19 RGC oies in the retin persist fter perio of xonopthy tht inlues erly efiits in The first two uthors ontriute eqully to this work n shoul therefore e regre s equivlent uthors. Corresponene: Toni S Rex, Deprtment of Ophthlmology n Visul Sienes, Vnerilt Eye Institute, Vnerilt University Meil Center, Lngfor MRB-IV, 2213 Grln Avenue, Nshville, Tennessee USA. Emil: toni.rex@vnerilt.eu Moleulr Therpy 1

2 EPO-R76E Protets Aginst Gluom The Amerin Soiety of Gene & Cell Therpy nterogre trnsport. 7,16 We report the effets of on xon trnsport, opti nerve histology, n vision. RESULTS Systemi gene elivery of EpoR76E oes not use n unsfe rise in hemtorit in the miroe olusion moel Mie in oth the pre- (59 ± 9%) n post-iop inution tretment groups (55 ± 4%) showe signifint inrese in hemtorit s ompre to mie tht reeive raav2/8.egfp (42 ± 3%; P <.1) or raav2/1.egfp (44 ± 2%; P <.1), respetively (Figure 1). The hemtorit level in oth ohorts ws within the norml rnge for mie. 2 In ll ses, the hemtorit Hemtorit (%) [EPO] (miu/ml) Figure 1 Systemi raav-meite elivery of EpoR76E uses rise in hemtorit n serum EPO onentrtion in miroe-injete mie. Hemtorit n serum EPO onentrtion were ssesse t olletion. () Box plot of hemtorit levels in mie tht reeive raav2/8.mepor76e n raav2/1.hepor76e ompre to mie tht reeive of the orresponing serotype. () Box plot of serum EPO onentrtion in mie tht reeive raav2/1.egfp or raav2/1.hepor76e. Dt re unville for mie tht reeive raav2/8.mepor76e ue to insuffiient sensitivity of the ELISA kit to mepo. EPO, erythropoietin; IOP, introulr pressure; raav, reominnt eno-ssoite virus. raav.hepor76e ws well elow tht inue y wil-type EPO, n phleotomy ws not neee. 9 Animls in the post-iop tretment group h signifintly inrese serum EPO onentrtion of 67 ± 28 miu/ ml s ompre to 2.4 ±.6 miu/ml in raav2/1.egfp-injete mie (P <.1; Figure 1). This inrese is similr to our previously pulishe stuies using this gene therpy strtegy. 7 Systemi gene elivery of EpoR76E oes not influene IOP elevtion in the miroe olusion moel Either 1 month following raav2/8.mepor76e ministrtion (pre-iop group) or immeitely prior to raav2/1.hepor76e ministrtion (post-iop group), IOP ws elevte y nterior hmer injetion of polystyrene miroes s esrie previously. 13 IOP ws elevte to n verge of 3.6 ±.3 mm Hg (verge ± SEM; P <.1) t 1 week post-injetion n remine stle over the ourse of the stuy (Figure 2). No signifint elevtion of IOP ws oserve in the sline-injete nimls. When strtifie y tretment group, we oserve no ifferene in IOP elevtion either initilly or over the 4-week perio etween nimls reeiving or (Figure 2 ), suggesting tht the effets of gene therpy re not ue to lowering IOP. This ws true whether ssessing solute vlue of IOP or hnge from seline (Figure 2 ). Both pre- n post-iop tretment with mintins norml RGC nterogre trnsport in the miroe olusion moel Ative nterogre xon trnsport of fluoresent holer toxin (CTB) from the RGC to the superior olliulus (SC) ws quntifie n onverte to retinotopi mp following previously pulishe methos. 17,21,22 In sline-injete mie, CTB ws trnsporte to ll retinotopi regions of the SC s expete (Figure 3). In ontrst, fol efiits in trnsporte signl were pprent in the -trete miroe-injete nimls (Figure 3). The pttern of this efiit progressing from the periphery to the representtion of the opti nerve is onsistent with previous finings. 23 These efiits were lrgely sent in oth the pre- n post-iop tretment groups (Figure 3,). Quntifition of intt trnsport throughout the SC volume showe 94 ± 5% n 75 ± 15% xon trnsport in -trete mie tht reeive n intrvitrel injetion of sline n miroes, respetively. Ative nterogre trnsport ws signifintly improve in oth the pre- n the post-iop tretment groups t 95 ± 6% n 9 ± 8%, respetively (P <.1; Figure 3e). Neither tretment group ws sttistilly ifferent from the sline-injete group, nor were the tretment groups sttistilly ifferent from eh other. Both pre- n post-iop tretment with preserves RGC xons in the miroe olusion moel In sline-injete eyes, the opti nerves ontine helthy xons s etermine y the ler xoplsm surroune y rkly stine myelin (Figure 4). In ontrst, 4 weeks fter miroe injetion, mny egenerting xon profiles were etete (Figure 4). Degenertive xons were ientifie y rk xoplsm n/or multi-lyering of myelin (Figure 4, rrows). 2

3 The Amerin Soiety of Gene & Cell Therpy EPO-R76E Protets Aginst Gluom IOP (mm Hg) IOP Chnge ( mm Hg) Time (weeks) Time (weeks) IOP (mm Hg) raav2/8.mepor76e raav2/8.egfp Sline Miroe raav2/8.egfp raav2/8.mepor76e 1 2 Time (weeks) IOP Chnge ( mm Hg) Time (weeks) raav2/1.hepor76e raav2/1.egfp raav2/1.egfp 3 4 raav2/1.hepor76e Figure 2 Introulr pressure is inrese in mie reeiving nterior hmer miroe injetion. () Grph of IOP level in miroe-injete mie ompre to sline-injete mie over 4-week perio following injetion (n = 6). () Grph of hnge in IOP in eh experimentl group over time. () Grph of hnge in IOP elevtion from seline over time in mie tht reeive raav2/8.mepor76e or raav2/8.egfp 1-month prior to miroe injetion (pre-iop). () Grph of hnge in IOP elevtion from seline over time in mie tht reeive raav2/1.hepor76e or raav2/1. egfp immeitely fter miroe injetion (post-iop). Arrows in n inite onset of gene expression from raav. IOP, introulr pressure; raav, reominnt eno-ssoite virus. Opti nerves from oth the pre- n post-iop tretment groups ppere to hve fewer egenertive xons thn the -injete mie (Figure 4,). The perentge of egenerting RGC xons ws 2.1 ± 1.2% n 1.8 ±.7% in the miroe-injete mie tht reeive pre- or post- IOP elevtion, respetively. Fewer egenerting xons were present in the opti nerves from miroe-injete mie tht reeive either pre- or post-iop elevtion; 1.2 ±.4% (P <.1) n 1.2 ±.6% (P <.5), respetively (Figure 4e). In ition, the groups showe unequl vrines of perent egenerting xons (P <.1). In the miroe, pre-iop, raav2/8. egfp mie, 43% of the opti nerves h 3.2 ±.9% egenerting xons (irle re), while the remining h 1.3 ±.2% egenerting xons (Figure 4e). In the miroe-injete mie reeiving raav2/1.egfp post-iop elevtion, 33% of the opti nerves h 2.6 ±.5% egenerting xons (irle re), while the remining h 1.3 ±.2% egenerting xons. This emonstrtes the presene of two popultions n illustrtes the vriility t the 4-week time point in this moel. In ontrst, only 1 (8%) n 2 (18%) opti nerves in the pre- n post-iop tretment groups, respetively, ontine greter thn 2% egenerting xons. No ifferene ws etete in the totl numer of xons etween groups. The totl numer of remining RGC xons ws 87 ± 16% n 88 ± 18% in the pre- n post-iop -trete mie, respetively, n 93 ± 16% n 96 ± 11% in the pre- n post-iop tretment groups, respetively (Figure 4f). Despite tren for erese loss of xons in the trete mie, the ifferene ws not sttistilly signifint, euse loss of xons in miroe eyes ws very mil. This is not entirely unexpete, sine in the miroe moel like other inuile moels, overt xon egenertion lgs efiits in trnsport n is sujet to vriility. 14,18,23 Vision is not signifintly erese t 4 weeks in the miroe olusion moel To ssess visul funtion, we performe flsh visul evoke potentils (fvep). The mplitue of the N1 n P1 peks (inite in Figure 5) represents the numer of funtionl RGC xons, while the lteny of the N1 n P1 represents how well the RGC xons propgte the tion potentil own the opti nerve. Men wveforms ompring seline n enpoint reorings showe n pprent efiit in N1 mplitue in the raav2/8. egfp miroe-injete group ut not the raav2/8.mepor76e (pre-iop) group (Figure 5). The P1 mplitue ppere erese t 4 weeks s ompre to seline (Figure 5). However, nlysis of the negtive re uner the urve of the fvep wveform from onset of stimulus to the pek of P1 showe no signifint ifferenes etween the raav2/8.egfp miroe-injete group (13 ± 47% of seline) n the raav2/8.mepor76e group (113 ± 43% of seline; Figure 5). In ition, while there Moleulr Therpy 3

4 EPO-R76E Protets Aginst Gluom The Amerin Soiety of Gene & Cell Therpy ppere to e tren towrs preservtion of the N1 n P1 mplitues, the ifferene i not reh sttistil signifine (Figure 5,). The N1 mplitue ws reue 14.5 ± 7.5% in the raav2/8.egfp miroe-injete group ompre to 1.3 ± 7.3% in the raav2/8.mepor76e-trete group (P vlue not signifint; Figure 5). There ws P1 mplitue reution of 21.2 ± 7.3% in the raav2/8.egfp miroe-injete group n 2.6 ± 8.6% in the raav2/8.mepor76e trete group (Figure 5), n the groups were not sttistilly ifferent from eh other (P =.98). Due to the sutle funtionl efiits oserve in the raav2/8. egfp miroe-injete nimls t this 4-week time point n lk of sttistil enefit from tretment with raav2/8.mepor76e given prior to onset of elevte IOP, we i not ssess fvep in the post-iop tretment group. raav2/8.mepor76e resulte in slight rise in hemtorit n h no effet on IOP elevtion in the DBA/2J moel Mie trete with raav2/8.egfp h hemtorit of 42 ± 1.9%, while those tht reeive raav2/8.mepor76e h n verge hemtorit of 48 ± 2.7% (P <.1; Figure 6). This level of inrese in hemtorit is omprle to tht inue in our previous stuies. 7 9 In ition, the hemtorit level in oth the raav2/8.egfp n raav2/8.mepor76e groups ws within the norml rnge for mie. 2 The IOP egn to inrese t 5 months of ge n y 7 months most of the mie h elevte IOP (Figure 6). The pek of gene expression from raav2/8 is 2 3 weeks fter trnsution. 24 Mie were trnsue with raav2/8 t 5 months of ge, resulting in therpeuti levels of gene expression t months. Tretment with raav2/8.mepor76e h no effet on IOP level (Figure 6). IOP mesurements were not performe pst 8 months of ge ue to ge-relte hnges to the orne tht n influene reings. 25,26 However, iret mesurement of IOP using nnultion inites tht mie with elevte IOP t 8 months of ge typilly ontinue to hve elevte IOP out to 1 months of ge. 17,27 In some eyes, IOP n erese t oler ges (e.g., months) ue to ongoing iris trophy. 19 The urrent stuy ws omplete t 1 months of ge, when most DBA/2J mie emonstrte nerly omplete loss of xon trnsport from the retin to the SC inepenent of IOP. 17 Post-IOP tretment with raav2/8.mepor76e preserve xon trnsport in the opti nerve in the DBA/2J moel Axon trnsport of CTB ppere fully intt in the 3-month-ol DBA/2J mie (Figure 7). In the 1-month-ol DBA/2J mie trete with raav2/8.egfp, there ws erese xon trnsport throughout the SC n pronoune setorl efiits, onsistent with previous oservtions in this strin (Figure 7). 28 In the 1-month-ol DBA/2J mie tht reeive raav2/8.mepor76e, xon trnsport ws iminishe s ompre to the 3-monthol ontrols, ut there ws no setorl efiit s in the raav2/8. egfp mie (Figure 7). The fluoresene in the SC ws quntifie to etermine the perent intt trnsport (Figure 7). The 3-month-ol mie exhiite 99 ±.8% intt trnsport. Consistent with the originl hrteriztion of this metho in these mie, there ws wie rnge in xon trnsport levels in e Perent intt trnsport (%) Figure 3 RGC nterogre trnsport is preserve 4 weeks fter miroe injetion in mie trete with. ( ) Representtive normlize fluoresene intensity het mps of the SC with re representing mximum intensity n lue/lk representing no fluoresene: () sline,, () miroe,, () miroe, raav2/8.mepor76e (pre-iop), n () miroe, raav2/1. hepor76e (post-iop tretment). The representtion of the opti nerve he is inite (sterisk). (e) Box plot of perent intt trnsport of mie injete with miroes n (n = 5), raav2/8. mepor76e (n = 8), or raav2/1.hepor76e (n = 9). IOP, introulr pressure; raav, reominnt eno-ssoite virus; SC, superior olliulus. the 1-month-ol raav2/8.egfp-trete mie. 17 Nerly hlf of the mie h no intt trnsport, few h lmost ompletely intt trnsport, n the remining hlf exhiite 5 8% trnsport (Figure 7). Every SC exmine in the 1-month raav2/8. mepor76e-trete group exhiite 43% or higher levels of xon trnsport, n there ws no exmple of zero trnsport. The verge perent intt xon trnsport in these mie ws 71 ± 17%. There ws sttistilly signifint ifferene in xon trnsport etween the 3-month n the 1-month raav2/8.egfp groups (P <.1) n etween the 1-month raav2/8.egfp n 1-month raav2/8.mepor76e groups (P <.5). There ws no sttistilly signifint ifferene etween the 1-month-ol raav2/8.mepor76e n the 3-month-ol ontrols. Post-IOP tretment with raav2/8.mepor76e preserve struture of the opti nerve in the DBA/2J moel The opti nerves from 1-month-ol mie trete with raav2/8. mepor76e looke omprle to those from 3-month-ol ontrol raav.hepor76e Sline Miroe 4

5 The Amerin Soiety of Gene & Cell Therpy EPO-R76E Protets Aginst Gluom e Degenerting xons (% of totl) f Totl xons 6, 4, 2, Sline Miroe Sline Miroe Figure 4 Opti nerve histology is preserve 4 weeks fter miroe injetion in mie trete with. ( ) Representtive right-fiel imges of p-phenyleneimine-stine opti nerve ross setions proximl to the retin of mie trete with: () sline,, () miroe,, () miroe, raav2/8.mepor76e (pre-iop), () miroe, raav2/1.hepor76e (post-iop). Arrows inite egenerting xons. Br = 25 µm. (e) Stter plot of the perent of egenerting xons in the opti nerves of mie from eh experimentl group. Cirles inite opti nerves with perent egenertion greter thn 1 SE ove the men. (f) Stter plot of totl xons in the opti nerves in the -trete miroe-injete mie (n = 22) n mie in the pre-iop (n = 13) n post-iop tretment (n = 11) groups. IOP, introulr pressure; raav, reominnt eno-ssoite virus. mie (Figure 8,). In ontrst, the opti nerves from 1-monthol mie trete with raav2/8.egfp ontine mny egenerting xons (Figure 8). Degenertive xons were ientifie y rk xoplsm n/or multilyering of myelin (Figure 8,, rrows). The numer of egenerting, live, n totl xons ws quntifie in eh group using mske, mnul, stnrize proeure. The perent of egenerting xons in 3-month-ol ontrols, 1-monthol raav2/8.egfp-, n raav2/8.mepor76e-trete mie ws.2 ±.1%, 7.7 ± 6.8%, n.8 ±.5%, respetively (Figure 8). The numer of live xons in 3-month-ol ontrols, 1-monthol raav2/8.egfp-, n raav2/8.mepor76e-trete mie ws 42,129 ± 4,124, 3,836 ± 14,923, n 5,293 ± 4,621, respetively (Figure 8e). The totl numer of xons in 3-month-ol ontrols, 1-month-ol raav2/8.egfp-, n -trete mie ws 42,195 ± 4,138, 32,551 ± 13,945, n 5,676 ± 4,43, respetively (Figure 8f). There ws sttistilly signifint hnge etween the 3-month n 1-month raav2/8.egfp group in eh se (P <.5). There ws lso sttistilly signifint ifferene Moleulr Therpy 5

6 EPO-R76E Protets Aginst Gluom The Amerin Soiety of Gene & Cell Therpy 25 1 µv 5 ms N1 P1 Bseline raav.repor76e Negtive re uner urve (% of seline) N1 mplitue (% of seline) 1 5 P1 mplitue (% of seline) 1 5 Figure 5 The fvep shows tren of improvement of funtionl vision 4 weeks fter miroe injetion in mie trete with raav2/8. mepor76e (pre-iop). () Overly of verge wveforms from mie prior to (seline) n 4 weeks fter miroe injetions n tretment with AAV2/8.eGFP (n = 13) or raav2/8.mepor76e (n = 7). The peks of the N1 n P1 mplitues re lele. () Box plot quntifition of the negtive re uner the urve from 2 to 175 ms (the verge P1 lteny) presente s perentge of seline. () Box plot of N1 mplitue presente s perent hnge from seline. () Box plot of P1 mplitue presente s perent hnge from seline. fvep, flsh visul evoke potentil; raav, reominnt eno-ssoite virus. etween the 1-month raav2/8.egfp n 1-month raav2/8. mepor76e mie in eh se (P <.1). There ws no ifferene etween the 3-month-ol ontrols n the 1-month raav2/8. mepor76e opti nerves in ny of the quntittive mesures. Post-IOP tretment with raav2/8.mepor76e preserves funtionl vision in the DBA2/J moel Averge wveforms showe erese in the fvep in 1-month-ol mie trete with raav2/8.egfp tht is somewht ttenute in nimls tht reeive raav2/8.mepor76e (Figure 9). Similr to our previous stuy, there ws no ifferene in the N1 or P1 lteny t 1 months s ompre to 3 months regrless of tretment (Figure 9). In ontrst, there were ifferenes in the N1 n P1 mplitues (Figure 9,). The verge N1 mplitue in the 3-month-ol mie ws 39 ± 7.9 µv (Figure 9). This ws erese to 24 ± 12 µv in the 1-month-ol raav2/8. egfp group. The N1 mplitue in the 1-month-ol raav2/8. mepor76e group ws 33 ± 8.9 µv. The perent of seline ws 6 ± 32% n 84 ± 22% in the raav2/8.egfp n raav2/8. mepor76e groups, respetively (P <.1). The verge 3-month P1 mplitue ws 49 ± 9.6 µv (Figure 9). This erese to 24 ± 15 µv n 35 ± 18 µv in the 1-month-ol raav2/8.egfp n raav2/8.mepor76e groups, respetively. The perent selines were 48 ± 31% n 7 ± 36% for the raav2/8.egfp n raav2/8.mepor76e groups, respetively. The ifferene etween the two groups ws sttistilly signifint (P <.5). For oth the N1 n P1 mplitue, there ws lso sttistilly signifint ifferene in vrine etween the groups (P <.5). This is likely ue to the known vriility in the evelopment of gluom symptoms y the DBA/2J. 19 DISCUSSION The ourse of neuroegenertion in gluom egins with efiits in tive xon trnsport followe y egenertion of the xons in the opti nerve n finlly eth of the RGCs. 18 In this stuy, we evlute the therpeuti potentil of EPO-R76E in two moels of gluom exhiiting very ifferent time ourses n severity of isese progression. This llowe for greter insight into the effetiveness of this tretment strtegy in this omplex linil isese. The suessful tretment of two moels of gluom is lso insightful in terms of mehnism. For exmple, while neuroinflmmtory proesses hve een shown to ffet RGC survivl following elevte pressure in vitro, the relevne to the isese proess in the miroe olusion moel n in gluom overll is not yet ler. 29 The role of neuroinflmmtion in the pthogenesis of gluom ppers to epen on the niml moel use, thus unersoring the nee to test potentil therpies in multiple moels. This is illustrte in the ility to ompletely lok neuroegenertion in the DBA/2J moel y meliorting peripherl immune response, while this sme pproh h no effet on lser photoogultion moel of oulr hypertension. 3,31 EPO n erese neuroinflmmtion n protet neurons y limiting ell eth (for review, see ref. 5). However, this oes not seem to e the relevnt mehnism in this stuy sine we etete protetion in the miroe olusion moel espite lk of 6

7 The Amerin Soiety of Gene & Cell Therpy EPO-R76E Protets Aginst Gluom 6 Hemtorit (%) IOP (mm Hg) Perent intt trnsport (%) Age (months) months 1 months Figure 6 Tretment of DBA/2J mie with raav2/8.mepor76e uses n elevtion in hemtorit, n no hnge in IOP s ompre to raav2/8.egfp. () Box plot of enpoint hemtorit in mie trete with raav2/8.egfp or raav2/8.mepor76e. () Grph of IOP over time in mie trete with raav2/8.egfp or raav2/8.mepor76e. Arrow inites onset of pek gene expression levels from raav. IOP, introulr pressure; raav, reominnt eno-ssoite virus. RGC eth t the erly time point nlyze. EPO n lso t on neuroinflmmtory proesses y loking infiltrtion or prolifertion of miroglil ells, or mroglil hypertrophy (for review, see ref. 5). Finlly, EPO n protet y eresing oxitive stress, n role for oxitive stress in gluom pthogenesis hs een reporte EPO tivtes Nrf2, whih inues trnsription of ntioxint enzymes from the ntioxint response element (for review, see ref. 5). Future stuies will investigte whih tivity of EPO is most relevnt to its neuroprotetive tion in gluom. In the miroe olusion moel, efiits in xon trnsport re first evient fter 2 4 weeks of elevte pressure. 17 In ition to the trnsport efiits, we lso etete smll numer of egenerting xons without hnge in the overll numer of xons, gin suggesting tht 4 weeks fter miroe injetion represents very erly stge in gluom. As preite y these results, we etete no sttistilly signifint efiit in the fvep, sine there is tremenous reunny t the level of the RGCs suh tht mny ells must e lost prior to the etetion of visul efiit. 36 As woul e preite se on the ove finings, our gretest eviene of neuroprotetion y ws from the xon trnsport mesurements, followe y quntifition of Figure 7 Tretment of DBA/2J mie with raav2/8.mepor76e t 5 months preserves RGC xon trnsport t 1 months. ( ) Representtive het mps of CTB fluoresene in the SC t () 3 months (n = 9) or t 1 months in mie trete with () raav2/8.egfp (n = 21) or () raav2/8.mepor76e (n = 19). () Stter plot of perent intt trnsport in 3-month-ol mie n 1-month-ol mie trete with raav2/8.egfp or raav2/8.mepor76e. Note the suset of SC with no evient xon trnsport in the raav2/8.egfp group, ut not in the raav2/8.mepor76e group. CTB, holer toxin ; raav, reominnt eno-ssoite virus; SC, superior olliulus. egenerting xons in the opti nerve. Despite the mil effets on the fvep, we still etete tren towr preservtion of the fvep in mie tht reeive. Importntly, similr to the niml moels, hnges in the opti is n elevte IOP re often etete in ptients prior to the evelopment of visul fiel efiits. 37 These ptients re given IOP-lowering tretments prior to vision loss, without etriment, n our results suggest tht this timing woul lso e sfe for EpoR76E gene therpy. Future stuies will etermine if extening the stuy out to 8 weeks of elevte pressure will result in sttistilly signifint loss in vision tht n e prevente y tretment with. In the DBA/2J, we ssesse therpeuti effiy t 1 months of ge, lte time point in the ourse of isese in this moel. At this ge, the opti nerve is severely egenerte, n mny RGCs hve een lost. 19 We previously trete DBA/2J mie t 1 month of ge with n etete omplete protetion of the RGC xons, ell oies, n vision (fvep) t 1 months. 7 In the urrent stuy, the level of protetion ws lower suggesting tht isese Moleulr Therpy 7

8 EPO-R76E Protets Aginst Gluom The Amerin Soiety of Gene & Cell Therpy e f 3 8, 8, 6, 6, Degenerting xons (% of totl) 2 1 Live xons 4, 2, Totl xons 4, 2, 3 months 1 months 3 months 1 months 3 months 1 months Figure 8 Tretment of DBA/2J mie with raav2/8.mepor76e t 5 months preserves RGC xon histology t 1 months. ( ) Representtive right fiel mirogrphs of opti nerves from () 3-month-ol mie (n = 11) or from 1-month-ol mie trete with () raav2/8.egfp (n = 15) or () raav2/8.mepor76e (n = 11). Degenertive xons re inite y rrows. Br = 25 µm. ( f) Stter plots of quntifition of () perent egenerting xons, (e) live xons, n (f) totl xons, irle inites ohort of highly egenerte opti nerves tht were not present in the other groups. raav, reominnt eno-ssoite virus 2 15 Time (ms) µv 5 ms 3 months 3 months 3 months N1 P1 N1 mplitue (% of seline) 1 5 % of seline Figure 9 Tretment of DBA/2J mie with raav2/8.mepor76e t 5 months prtilly preserves the fvep t 1 months. () Overly of verge fvep wveforms from 3-month-ol mie (n = 4) n 1-month-ol mie trete with raav2/8.egfp (n = 24) or raav2/8.mepor76e (n = 24). () Box plot of verge N1 n P1 ltenies from ll groups. (,) Box plots of the () N1 n () P1 mplitues in the two 1-month groups presente s perent seline (3 month). Only two (8%) points re elow 5% of N1 seline in the raav2/8.mepor76e group s ompre to 1 (42%) in the raav2/8.egfp group. fvep, flsh visul evoke potentil; raav, reominnt eno-ssoite virus. 8

9 The Amerin Soiety of Gene & Cell Therpy EPO-R76E Protets Aginst Gluom proesses h lrey strte n oul not e ompletely stoppe y EPO-R76E. It is unler from this stuy whether tretment with loke the onset of egenertive pthwys in neurons jent to egenertive neurons or slowe the rte of egenertion in ll neurons. If the ltter is true, then it suggests tht espite the pleiotropi nture of EPO, it oes not t on ll egenertive proesses tht re initite y rise in IOP. Finlly, it is possile tht the erese effiy of when elivere t 5 months s oppose to 1 month in the DBA/2J my e ue, in prt, to egenertive proesses ourring in these mie inepenent of gluom. For exmple, mirogli numers n retivity re elevte in the retin 3 months efore elevtion of IOP in this moel. 38 Also, these mie unergo photoreeptor egenertion, whih is likely to e unrelte to the evelopment of gluom sine efiits re etetle prior to elevtion of IOP. 39 Future longer-term stuies in the miroe olusion moel will help to nswer some of these questions. This stuy provies eviene tht elivery of, with linilly relevnt timing (t onset of elevte IOP), limits the mount of egenertion n vision loss in two well-reognize moels of gluom. 4 This tretment prigm is vntgeous euse long-term therpy is provie from single injetion without ngerous rise in hemtorit, mking it sfer n more linilly vile. 7 9 MATERIALS AND METHODS Virl vetor proution. Muttion R76E ws rete in rhesus mque Epo gene (mepor76e) n humn Epo gene (hepor76e) vi site-irete mutgenesis n lone into n AAV2 genome plsmi uner the ontrol of the CMV promoter. Both vetors were pkge n purifie y the University of Pennsylvni Vetor Core (Philelphi, PA). Cpsi protein plsmi from AAV serotype 8 ws use with mepor76e to rete hyri serotype raav2/8.cmv.repor76e (use for pre-iop tretment in the miroe olusion moel, n for ll DBA/2J stuies), n psi protein plsmi from AAV serotype 1 ws use with hepor76e to rete hyri serotype raav2/1.cmv.hepor76e (use for post-iop tretment in the miroe olusion moel). The titers were g/ml for raav2/8.cmv.mepor76e n g/ml for raav2/1.cmv. hepor76e. Stok raav2/8.cmv.egfp vetor ws purhse from the University of Pennsylvni Vetor Core. Mie. All niml experiments were onute in ompline with the ARVO Sttement for the Use of Animls in Ophthlmi n Vision Reserh. This stuy ws rrie out uner n niml protool pprove y the Vnerilt University Meil Center Institutionl Animl Cre n Use Committee. DBA/2J n C57BL/6J mie were otine from Jkson Lortories (Br Hror, ME). For the miroe olusion moel, only mle mie ~3 4 months of ge were utilize for miroe injetion to ontrol for ge- n gener-epenent vriility. Mie were re in-house, mintine on iurnl light yle, n provie foo n wter liitum. raav injetions. C57BL/6J mie reeive single 1 µl injetion of g of vetor in the right qurieps. For tretment eginning prior to onset of elevte IOP (i.e., Pre-IOP), mie reeive raav2/8.cmv.mepor76e or raav2/8.cmv.egfp 1 month efore miroe injetion sine it tkes ~3 weeks to reh pek gene expression from this serotype. 24 For tretment eginning t onset of elevte IOP (i.e., post-iop), mie reeive raav2/1. CMV.eGFP or raav2/1.cmv.hepor76e immeitely fter miroe injetion to result in pek trnsgene expression 1 week lter. 24 All mie were ollete 1-month post-miroe injetion, i.e., pretretment mie were ollete 2 months post-raav injetion, while post-elevte IOP tretment mie were ollete 1-month post-raav injetion. DBA/2J mie reeive single 1 µl injetion of g of raav2/8.cmv.mepor76e or raav2/8.cmv.egfp s ontrol to the right qurieps t 5 months of ge to result in pek gene expression 3 weeks lter. 41 Mie were euthnize t 1 months of ge. Miroe olusion moel. Inution of IOP elevtion ws inue ilterlly vi injetion of 15-µm imeter FluoSpheres polystyrene miroes (Thermo Fisher, Wlthm, MA) into the nterior hmer s esrie previously for mie. 13,14 Briefly, 1.5 mm outer imeter/1.12 mm inner imeter filmente pillry tues (Worl Preision Instruments, Srsot, FL) were pulle using P-97 horizontl puller (Sutter Instrument Compny, Novto, CA), n the resulting neeles were roken using foreps to n inner imeter of ~1 µm. Miroes (or ltte Ringer s sline solution s ontrol) were loe n injete using miroinjetion pump (Worl Preision Instruments, Srsot, FL). Mie were nesthetize with isoflurne n ilte using topil 1% tropimie ophthlmi solution (Ptterson Veterinry, Devens, MA), n 2 µl (~2, miroes) were injete. The neele ws mintine in the injetion site for 2 seons efore retrtion to reue miroe efflux. Mie were given topil.3% tormyin ophthlmi solution (Ptterson Veterinry, Devens, MA) following injetion. IOP mesurement. IOP ws mesure using the Ire TonoL reoun tonometer (Colonil Meil Supply, Frnoni, NH). Mie were nesthetize using isoflurne, n 1 mesurements were quire from eh eye within 2 minutes of inution of nesthesi. IOP ws mesure in mie immeitely prior to miroe injetion n weekly following miroe injetion. In the DBA/2J, IOP ws mesure t 3 months (n = 26, raav2/8. egfp; n = 52 raav2/8.mepor76e) n then twie month from 5 8 months (n = 42 52). If no elevtion in IOP ws etete y 8 months, the eye ws exlue from stuy. Tissue olletion, hemtorit, n serum EPO quntifition. Bloo ws ollete y ri punture prior to perfusion. Hemtorit ws etermine my pillry tue entrifugtion using the CritSpin system (Bekmn Coulter, Bre, CA). EPO onentrtion ws mesure y snwih ELISA using Quntikine Humn EPO ELISA kit (R&D Systems, Minnepolis, MN) oring to mnufturer iretions. Animls were then perfuse with 4% prformlehye, n eyes, opti nerves, n rins were ollete n ple in 4% prformlehye. Retinl gnglion ell nterogre trnsport tring. Mie were nesthetize with isoflurne n injete intrvitrelly with 2 µl CTB onjugte to Alex Fluor 594 (Thermo Fisher, Wlthm, MA) using 3 guge Hmilton syringe. Approximtely 3 ys following injetion, mie were nesthetize vi intrperitonel injetion of 2,2,2-triromoethnol (Sigm-Alrih, Sint Louis, MO) n perfuse with 4% prformlehye in phosphte-uffere sline. Brins were postfixe in 4% prformlehye in phosphte-uffere sline, infiltrte with 3% surose, n ryosetione oronlly t 5 µm. Setions trversing the SC were mounte, n the SC ws imge vi epifluoresene mirosopy (Nikon Instruments, Melville, NY). Fluoresene in the SC setions ws quntifie using ImgePro softwre (Mei Cyernetis, Rokville, MD) using n utomte mro esrie erlier to quntify the frtion of the SC retinotopi representtion ontining intt nterogre trnsport. 17,21,22 Briefly, fluoresene intensity ws summe long the vertil orsl ventrl olumns through the SC setions, n normlize fluoresene intensity sore ws generte long the meil lterl length of the SC. A fluoresene intensity het mp ws generte y omining ll setions trversing the SC. This resulte in omplete retinotopi mp, with re representing mximum fluoresene n lue representing no fluoresene. Intt trnsport is efine s >7% of mximum fluoresene. Opti nerve histology. Setions of the myelinte opti nerve ~1 mm ehin the gloe were postfixe in 1% glutrlehye n 4% prformlehye in Moleulr Therpy 9

10 EPO-R76E Protets Aginst Gluom The Amerin Soiety of Gene & Cell Therpy phosphte-uffere sline for t lest 24 hours. Nerves were further postfixe in 2% osmium tetroxie for 1 hour n emee in low visosity Spurr s emeing mei (Eletron Mirosopy Sienes, Htfiel, PA). Semi-thin 7-nm setions were ut n stine with 1% p-phenyleneimine. Setions were imge using right fiel mirosopy with 1 oil-immersion ojetive (Nikon Instruments, Melville, NY). Axons were mnully ounte using ImgeJ softwre y smpling 2% of the totl nerve ross-setionl re using fixe gri overly to estimte xon ensity in the nerve (xons/mm 2 ). Totl numer of surviving xons ws estimte s the prout of men xon ensity n nerve ross-setionl re, s esrie previously. 25 Flsh visully evoke potentil. Mie were rk-pte overnight. Mie were nesthetize y intrperitonel injetion of 25/8/6 µg/g oy weight of ketmine, xylzine, n urethne, n eyes were ilte with 1% tropimie. After plement in hete Gnzfel ome (Dignosys, Lowell, MA), pltinum eletroes (Grss Tehnologies, West Wrwik, RI) were ple suermlly ove the visul ortex 3 mm lterl from the miline, n referene eletroe ws ple suermlly in the snout. Mie were presente with white light flshes 1. -s/m 2 t frequeny of 1 Hz n n inter-sweep ely of 5 ms. Resulting wveform n mplitues were the verge of 2 sweeps. Negtive re uner the urve of the VEP wveform ws quntifie etween n 175 ms (the verge P1 lteny). Sttistil nlysis. All results re represente s verge ± SD unless otherwise inite. For IOP mesurement t, two-wy nlysis of vrine ws use. For the miroe olusion moel stuies, one-wy nlysis of vrine ws use, employing the Dunnett post-ho test for pirwise omprison. Comprisons were only me etween the n groups, not etween tretment groups. For the DBA/2J moel stuies, one-wy nlysis of vrine ws use, employing the Tukey post ho test. Vrines were lulte using the Brown Forsythe test. Signifine ws ssume t P <.5. In ll figures, the following initors of sttistil signifine re use: P <.5; P <.1; P <.1. ACKNOWLEDGMENTS This reserh ws fune y NIH grnts R1 EY22349 (T.S.R.), T32 EY (W.S.B.), n P3 EY8126 (D.J.C.), Deprtment of Defense grnt W81XW , the Gluom Reserh Fountion (D.J.C.), Reserh to Prevent Blinness Unrestrite Funs (Pul Sternerg, Jr.), n Fight for Sight Summer Reserh Fellowship (Y.L.G.). The uthors thnk Lorrine Ksml for ssistne in xon quntifition. T.S.R. is o-inventor in pening US ptent pplition (13/979,451) n interntionl ptent pplition (PCT/212/21247) regring neuroprotetive use of EPO-R76E. No other uthors hve onflits of interest to islose. Referenes 1. Shwrtz, K n Buenz, D (24). Current mngement of gluom. Curr Opin Ophthlmol 15: Olthoff, CM, Shouten, JS, vn e Borne, BW n Weers, CA (25). Nonompline with oulr hypotensive tretment in ptients with gluom or oulr hypertension n eviene-se review. Ophthlmology 112: Broxmeyer, HE (213). 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