original article 954 vol. 17 no. 6, june 2009 Genzyme Corporation, Framingham, Massachusetts, USA

Transcription

1 originl rticle The Americn Society of Gene Therpy Glycoengineered Acid α-glucosidse With Improved Efficcy t Correcting the Metolic Aerrtions nd Motor Function Deficits in Mouse Model of Pompe Disese Yunxing Zhu 1, Ji-Lei Jing 1, Nthn K Gumlw 1, Jinhu Zhng 1, Scott D Bercury 1, Roin J Ziegler 1, Kren Lee 1, Mriko Kudo 1, Willim M Cnfield 1, Timothy Edmunds 1, Cnwen Jing 1, Roert J Mttlino 1 nd Seng H Cheng 1 1 Genzyme Corportion, Frminghm, Msschusetts, USA Improving the delivery of therpeutics to disese-ffected tissues cn increse their efficcy nd sfety. Here, we show tht chemicl conjugtion of synthetic oligoscchride hroring mnnose 6-phosphte (M6P) residues onto recominnt humn cid α-glucosidse (rhgaa) vi oxime chemistry significntly improved its ffinity for the ctionindependent mnnose 6-phosphte receptor (CI-MPR) nd susequent uptke y muscle cells. Administrtion of the crohydrte-remodeled enzyme (oxime-neo-rhgaa) into Pompe mice resulted in n pproximtely fivefold higher clernce of lysosoml glycogen in muscles when compred to the unmodified counterprt. Importntly, tretment of immunotolerized Pompe mice with oximeneo-rhgaa trnslted to greter improvements in muscle function nd strength. Treting older, symptomtic Pompe mice lso reduced tissue glycogen levels ut provided only modest improvements in motor function. Exmintion of the muscle pthology suggested tht the poor response in the older nimls might hve een due to reduced regenertive cpcity of the skeletl muscles. These findings lend support to erly therpeutic intervention with trgeted enzyme s importnt considertions in the mngement of Pompe disese. Received 6 Jnury 9; ccepted Ferury 9; pulished online 1 Mrch 9. doi:1.138/mt.9.37 Introduction Mnipulting the crohydrte moieties on therpeutic drugs to improve their iologicl chrcteristics is gining interest s generl strtegy to increse their overll performnce. Over the pst severl yers, glycoengineering hs een successfully deployed to enhnce the phrmcokinetics nd phrmcodynmics of severl glycoprotein iologics. 1 For exmple, the introduction of n dditionl site of N-linked glycosyltion onto recominnt erythropoietin ws effective in generting modified iologic with incresed circulting hlf-life nd therey extended iologicl ctivity. 2 Anlogous mnipultion of tissue plsminogen ctivtor to reduce the numer of glycosyltion sites ws lso successful in generting longer-cting vrint. 3 Improved trgeting of glycoproteins to disese-ffected cells nd orgns could lso e relized through ltertion of their crohydrte composition. A prototypicl exmple would e the enzymtic remodeling of the crohydrte on recominnt glucocererosidse to expose the core mnnose residues on its oligoscchride side chins. This mneuver enhnced its recognition nd uptke y the mnnose receptor on mcrophges, the cells tht re primrily ffected in Gucher disese. 4, As n extension of these findings, we sought to evlute the reltive merits of engineering the crohydrte on recominnt humn cid α-glucosidse (rhgaa) s n pproch to increse its delivery to the muscles of sujects with Pompe disese. Acid α-glucosidse (GAA) is lysosoml enzyme tht is involved in the norml ctolism of glycogen, the deficiency of which cuses Pompe disese. 6,7 A reduction or loss of GAA ctivity results in the progressive ccumultion of lysosoml glycogen in vriety of cells, with the myocytes of the crdic, respirtory, nd skeletl muscles eing most severely ffected. Depending on the extent of enzyme deficiency, sujects with Pompe disese present rod spectrum of clinicl symptoms rnging from severe hypotoni, generlized muscle wekness, nd profound hypertrophic crdiomyopthy in infntile ptients 8,9 to miniml crdic involvement in lte-onset ptients Ptients with the infntile form of the disese invrily die ~1 yer of ge from crdic filure. In the lte-onset ptients, progressive deteriortion of the respirtory nd skeletl muscles leds to significnt moridity nd in mny instnces erly mortlity, generlly from respirtory filure. Significnt efforts hve een pplied towrd developing n enzyme replcement therpy for Pompe disese culminting with the recent regultory pprovl of rhgaa produced from Chinese hmster ovry cells for treting the infntile popultion The clinicl experience with enzyme replcement therpy indicted tht dministrtion of mg/kg Correspondence: Seng H Cheng, Genzyme Corportion, 49 New York Avenue, Frminghm, Msschusetts , USA. E-mil: seng.cheng@genzyme.com or Yunxing Zhu, Genzyme Corportion, 49 New York Avenue, Frminghm, Msschusetts , USA. E-mil: yunxing.zhu@genzyme.com 94 vol. 17 no. 6, june 9

2 The Americn Society of Gene Therpy Second Genertion Enzyme for Pompe Disese of rhgaa is effective t ddressing severl of the disese mnifesttions. However, correction of the pthology in the skeletl muscles ppers to present chllenge, prticulrly in ptients with more dvnced disese tht could not e resolved with higher doses or more frequent infusions of the enzyme. In prt, this refrctoriness my e due to the reltively low efficiency y which the enzyme cn e trnslocted from the circultory system cross the endothelil cells nd interstitil tissues to the ffected muscles. The ction-independent mnnose 6-phosphte receptor (CI-MPR) on skeletl muscle tht is primrily involved in the cellulr uptke of rhgaa is lso reportedly present in low undnce in this tissue. 16,17 To ddress this potentil limittion of enzyme replcement therpy, we sought to improve the delivery of rhgaa to the ffected muscles. Uptke of exogenous rhgaa y the skeletl muscles is medited primrily y the CI-MPR. 18 However, nlysis of rhgaa prepred from different sources including those from Chinese hmster ovry cells or the milk of trnsgenic rits indicted tht they hrored only modest levels of the cognte lignd, mnnose 6-phosphte (M6P). 19 Introduction of dditionl M6P moieties onto rhgaa either y enzymtic engineering (HP-GAA) or chemicl conjugtion of synthetic oligoscchrides ering M6P residues (neo-rhgaa) hs een shown to improve their inding to the CI-MPR nd susequent uptke y cells in culture Moreover, in the cse of neo-rhgaa, dministrtion of the modified enzyme to Pompe mice resulted in greter clernce of the errnt ccumultion of lysosoml glycogen when compred with the unmodified enzyme. 21 We hve extended these findings nd report here our efforts to improve the chemicl stility of neo-rhgaa nd importntly, to scertin the effects of this modifiction on its iologicl ctivity. We show tht using oxime chemistry to conjugte synthetic oligoscchrides ering M6P residues onto rhgaa generted modified enzyme (oxime-neo-rhgaa) with higher stility in vivo nd n improved ility to reduce the urden of glycogen storge in the skeletl nd crdic muscles of Pompe mice. Importntly, we show tht ssocited with these iochemicl improvements were significnt enhncements in motor function in the treted Pompe mice. Results Genertion of conjugtes of rhgaa nd synthetic M6P-ering oligoscchrides with enhnced stility nd ffinity for the CI-MPR Previously, we hd reported the fesiility of conjugting synthetic oligoscchrides ering M6P residues (Figure 1) onto rhgaa using cronyl-coupled hydrzone chemistry.,21 Administrtion of the crohydrte-modified enzyme (neo-rhgaa) into Pompe mice resulted in higher uptke y the ffected muscles with susequent greter clernce of the ccumulted lysosoml glycogen compred to the unmodified, prent enzyme. Here, we evluted n lternte cronyl-coupled chemistry to generte n oxime ond using n minooxy-derived glycn. To ssess the reltive merits of using n oxime ond in the conjugte, the synthetic M6P-ering oligoscchride (Figure 1) contining hydrzide rective group ws first converted to n minooxy rective group through single step rection with N-(t-BOC)-minooxycetic cid tetrfluorophenyl ester, followed α1,2 M6P Mn α1,6 Mn α1,2 α1,3 M6P Mn OH OH OH O CH CH CH 2 Oxidtion Periodte α1,6 O O β Mn O (CH 2 ) 3 C NHNH C CH 2 ONH 2 C H Conjugtion + NH 2 O x M6P GAA GAA GAA N O x M6P C H Figure 1 Structure of the synthetic oligoscchride lignd nd scheme for its conjugtion to rhgaa. () Design of the synthetic oligoscchride used in the conjugtions studies. () Scheme used to conjugte the synthetic glycn to rhgaa. The silic cids on the enzyme were oxidized with periodte efore recting with the rective group (minooxy) on the synthetic glycn to generte oxime-neo-rhgaa. Symols in digrm: open circle, mnnose; open squre, N-cetylglucosmine; open dimond, glctose; open tringle, silic cid. GAA, cid α-glucosidse; M6P, mnnose 6-phosphte; rhgaa, recominnt humn cid α-glucosidse. y deprotection in trifluorocetic cid. The purified M6P-minooxy lignd ws then coupled to periodte-oxidized rhgaa s outlined in Figure 1. The conjugtion rection ws very efficient nd proceeded to completion when 4 μmol/l (~ mg/ ml) oxidized rhgaa ws rected with.7 mmol/l (1 mg/ ml) M6Pcontining glycn. Consistent with the ddition of M6P-ering lignds, the resultnt product, oxime-neo-rhgaa, demonstrted n incresed moleculr mss nd decresed isoelectric point due to the incorportion of dditionl phosphte groups (dt not shown). Susequent nlysis y Dionex chromtogrphy (Dionex, Sunnyvle, CA) indicted significnt increse in M6P content in the conjugted enzyme. Bsed on the numer of M6P on oximeneo-rhgaa nd the numer of silic cids on rhgaa, it would suggest tht synthetic glycns were ppended onto ll the silic cids on rhgaa. The oserved increse in moleculr weight of oxime-neo-rhgaa (~8 kd when compred with the unmodified enzyme) mesured using mss spectrometry or sodium dodecyl sulfte-gel electrophoresis (dt not shown) ws consistent with this ssumption. Mesurement of the enzymtic ctivity of oxime-neo-rhgaa showed n incresed K m (~%) presumly due to steric hindrnce y the dded oligoscchride side chins. However, fter the modified enzyme ws delivered to the lysosoml comprtment where the glycns were processed, its enzymtic ctivity ws similr to tht of unmodified rhgaa (dt not shown). The V mx of the modified enzyme ws not ffected. Although our preliminry studies showed tht the ntiody titers to dministrtion of oxime-neo-rhgaa nd unmodified enzyme were not different (dt not shown), further nlysis will e required to fully explore the repertoire of ntiodies generted ginst the two enzymes. Incresing the content of M6P residues on oxime-neorhgaa improved its ffinity for the CI-MPR when compred to the unmodified enzyme. Greter thn 9% of oxime-neorhgaa ound to CI-MPR column s compred to 1 3% of Moleculr Therpy vol. 17 no. 6 june 9 9

3 Second Genertion Enzyme for Pompe Disese The Americn Society of Gene Therpy the unmodified rhgaa (Figure 2). The ddition of M6P onto rhgaa lso significntly improved enzyme uptke y L6 myolsts in vitro (Figure 2), similr to those reported previously for enzyme conjugtes formed using hydrzone chemistry. Stility studies indicted tht those formed using oxime chemistry hd the dded dvntge of eing significntly more stle (Figure 2c). No mesurle dissocition of the synthetic glycn from oximeneo-rhgaa ws detected fter incution of the glycoconjugte t 2 C for 14 dys or 4 C for 9 dys. Together, these dt indicte GAA ctivity (RU) GAA ctivity ( 1, RU) c % Of strting M6P ,7 3, 2,7 2, 1,7 1, oxime-neo-rhgaa rhgaa Frctions rhgaa oxime-neo-rhgaa GAA concentrtion (nmol/l) 2 C 4 C Incution time (dys) Figure 2 Biochemicl chrcteristics of the unmodified nd glycoengineered enzymes. () Chromtogrphy of oxime-neo-rhgaa (open circle) nd rhgaa (closed circle) over CI-MPR column. Approximtely μg of the different enzymes were loded onto 2 ml column. After wshing the column with inding uffer, the ound mteril ws eluted (strting t frction 11) with inding uffer contining mmol/l M6P. Frctions (2 ml) were collected nd ssyed for GAA ctivity. () Uptke of the enzymes y L6 myolsts in culture. Incresing mounts of either oxime-neo-rhgaa (open circle) or rhgaa (closed circle) were dded to L6 myolsts nd incuted t 37 C for 18 hours. After wshing, the cells were lysed nd the enzyme ctivity in the lystes ssyed using the fluorogenic sustrte, 4-methylumelliferyl-α-d-glucopyrnoside. Enzyme ctivity ws expressed in reltive units (RU). (c) Stility of oxime-neo-rhgaa t 4 C (open squre) nd 2 C (closed tringle). The modified enzyme ws incuted t the respective tempertures for the times indicted fter which they were sujected to nlysis to determine the level of M6P. CI-MPR, ction-independent mnnose 6-phosphte receptor; GAA, cid α-glucosidse; M6P, mnnose 6-phosphte; rhgaa, recominnt humn cid α-glucosidse. tht oxime-neo-rhgaa hs the desired chrcteristics tht would support its doption for preclinicl development. Oxime-neo-rhGAA demonstrtes greter efficcy thn unmodified rhgaa t reducing glycogen storge in muscles of Pompe mice To scertin whether the improved chrcteristics of oxime-neorhgaa oserved in vitro trnslted to greter efficcy in vivo, -month-old Pompe mice were dministered four weekly doses of either the modified or unmodified enzyme. The muscles of Pompe mice t months of ge exhiit significnt lysosoml ccumultion of glycogen. Anlysis of the tissues 2 weeks fter the lst enzyme dministrtion showed tht oth versions of rhgaa reduced the levels of glycogen in the hert, diphrgm, qudriceps, nd triceps in dose-dependent mnner (Figure 3). However, tretment with oxime-neo-rhgaa ws more effective s illustrted y the oservtion tht comprle reduction in glycogen could e Glycogen (mg/g tissue) Hert Qudriceps i ii Sline mg/kg rhgaa mg/kg rhgaa 1 mg/kg rhgaa 1 mg/kg oxime-neo-rhgaa mg/kg oxime-neo-rhgaa Hert Diphrgm Qudriceps Triceps iii iv Vehicle mg/kg rhgaa mg/kg oximeneo-rhgaa Figure 3 Reltive ilities of unmodified nd modified rhgaa to reduce tissue glycogen levels in young Pompe mice. () Cohorts of -month-old Pompe mice were dministered incresing mounts of either rhgaa or oxime-neo-rhgaa. The mice (8 nimls/group) were treted with four weekly doses of enzyme nd killed 2 weeks fter the lst tretment. Tissues were collected nd ssyed for glycogen levels using the Amplex Red glucose ssy. Dt re expressed s mens ± SD. () Sections of the hert nd qudriceps were stined with PAS nd then nlyzed y high resolution light microscopy. Representtive sections from the hert of Pompe mice treted with vehicle (i), mg/ kg rhgaa (iii) nd mg/kg oxime-neo-rhgaa (v) re shown, s re qudriceps of nimls treted with vehicle (ii), mg/kg rhgaa (iv) nd mg/ kg oxime-neo-rhgaa (vi). Glycogen is visulized s purpleeded structures within the myocytes. PAS, periodic cid-schiff; rhgaa, recominnt humn cid α-glucosidse. v vi 96 vol. 17 no. 6 june 9

4 The Americn Society of Gene Therpy Second Genertion Enzyme for Pompe Disese ttined using fivefold lower dose of the modified enzyme. For exmple, the extent of glycogen clernce chieved using 1 nd mg/kg of oxime-neo-rhgaa ws comprle to tht ttined using nd 1 mg/kg of rhgaa, respectively (Figure 3). The greter efficcy of oxime-neo-rhgaa t reducing tissue glycogen content could lso e visulized y exmining tissue sections stined for glycogen with periodic cid-schiff regent (Figure 3, pnels iii nd iv). Anlysis of the hert nd qudriceps of nimls dministered mg/kg rhgaa showed significnt decrese in periodic cid-schiff stined structures compred to these sme tissues in untreted mice. However, mice treted with similr dose of oxime-neo-rhgaa showed n even more complete clernce of glycogen-contining structures from oth tissues (Figure 3, pnels v nd vi). Motor function improvement ws significntly greter in oxime-neo-rhgaa-treted immunotolernt Pompe mice To determine if the greter ility of oxime-neo-rhgaa to cler lysosoml glycogen deposits from the muscle trnslted to improved motor function, Pompe mice were lso sujected to vriety of functionl tests. Becuse improvements in muscle function could only e visulized over period of months nd ecuse weekly dministrtions of rhgaa engendered roust immune response to the humn enzyme, Pompe mice were first immunotolerized to rhgaa. This ws ccomplished y treting the nimls with recominnt deno-ssocited virus (AAV) vector encoding n inctive mutnt of rhgaa (D44N) under the trnscriptionl control of liver-restricted promoter. 22 This pproch of using AAV vectors to confer immunotolernce hs een reported for numer of different trnsgene products including GAA Intrvenous dministrtion of recominnt AAV vector encoding the mutnt enzyme (AAV8/DC19-GAA D44N ) into Pompe mice resulted in high-level heptic expression nd secretion of mutnt GAA D44N into the systemic circultion. However, s the specific ctivity of the mutnt GAA ws <1% of wild type GAA, there ws no mesurle decrese in glycogen levels in the Pompe-ffected tissues (Figure 4) compred to nive Pompe mice (dt not shown). One month fter dosing mice with AAV8/ DC19-GAA D44N, susequent periodic intrvenous dministrtions of rhgaa did not elicit ntiodies ginst the recominnt enzyme (dt not shown). In contrst, dministrtion of rhgaa into Pompe mice tht hd not received AAV8/DC19-GAA D44N developed high titers of ntiody to the hydrolse. To determine the reltive ility of the modified nd unmodified rhgaa to correct the motor function deficits of Pompe mice, the nimls were first dministered AAV8/DC19-GAA D44N when they were 4. months old. One month lter, fter the nimls were immunotolerized to the humn enzyme, cohorts of 1 mice ech were treted iweekly with mg/kg rhgaa, 1 mg/kg rhgaa, mg/kg oxime-neo-rhgaa, or vehicle for 8 months. To evlute the impct of the different tretments on muscle strength nd motor coordintion, the nimls were tsked with performing on rotrod progrmmed to rock ckwrds nd forwrds (referred to s rocking rotrod). In this ssy, the rotrod ws progrmmed to ccelerte to 17. rpm within 2. seconds fter which the direction of rottion ws reversed nd this repeted over period of Glycogen (mg/g tissue) Ltency (seconds) c Ltency (seconds) Vehicle mg/kg rhgaa 1 mg/kg rhgaa mg/kg oxime-neo-rhgaa Hert Diphrgm Qudriceps Triceps *** 6 seconds. In contrst to wild type mice, vehicle-treted Pompe mice displyed deficit in this ssy t the outset (4. months old) tht ecme grdully worse s the nimls ged (Figure 4). This ws indicted y the progressively shorter time the vehicletreted Pompe mice were le to remin on the rotrod. Pompe mice treted iweekly with mg/kg rhgaa showed trend towrd improvement ut this did not rech sttisticl significnce ** *** ++ *** +++ *** *** *** *** *** *** *** Time of tretment (months) Figure 4 Assessment of motor coordintion nd muscle strength fter enzyme therpy of young Pompe mice. Pompe mice (4. months old) were first dministered E11 drp of AAV8/DC19-GAA D44N to induce immunotolernce to humn GAA. One month fter dosing with the virl vector, the mice were treted iweekly with injections of different doses of rhgaa or oxime-neo-rhgaa for 8 months. () At the end of the study, the mice were killed nd their tissues nlyzed for glycogen levels. Dt re expressed s mens ± SD (n = 1 nimls per group). Throughout the study, the nimls (oth wild type nd Pompe mice) were sujected to () rocking rotrod nd (c) wire-hng tests. Age-mtched wild type mice (closed squre) nd Pompe mice treted with vehicle (closed circle), mg/kg rhgaa (closed tringle), 1 mg/ kg rhgaa (closed dimond), nd mg/kg oxime-neo-rhgaa (open circle) were tested monthly. Sttisticl nlyses were performed etween vehicle nd enzyme-treted groups (vehicle versus mg/kg oxime-neorhgaa, *P <., **P <.1, nd ***P <.1; vehicle versus 1 mg/ kg rhgaa, + P <., ++ P <.1, +++ P <.1), s well s etween oxime-neo-rhgaa- nd rhgaa-treted groups ( P <.1)) for ech time point. Dt re presented s mens ± SEM. drp, DNse resistnt prticle; rhgaa, recominnt humn cid α-glucosidse Moleculr Therpy vol. 17 no. 6 june 9 97

5 Second Genertion Enzyme for Pompe Disese The Americn Society of Gene Therpy when compred with the vehicle-treted cohort (Figure 4). Mice treted t the higher dose of 1 mg/kg rhgaa showed mrked improvement in motor function tht reched sttisticl significnce when compred to the vehicle-treted group fter months of enzyme therpy. The cohort dministered mg/kg oxime-neorhgaa fred the est, exhiiting sttisticlly significnt improvements over control nimls fter just 2 months on enzyme therpy nd reching levels of performnce tht were similr to wild type mice fter 8 months of therpy (Figure 4). At 8 months posttretment, the performnce of the cohort dministered mg/kg oxime-neo-rhgaa ws lso significntly greter thn tht treted with 1 mg/kg of unmodified enzyme. These improvements in motor function were consistent with the extent of glycogen clernce ttined using the different enzyme entities (Figure 4), nd indicted tht the modified oxime-neo-rhgaa ws t lest fivefold more efficcious thn the unmodified version t ddressing the motor function deficits in the Pompe mice. The enhnced therpeutic effect oserved with oxime-neorhgaa ws further corroorted y the wire-hng ssy tht mesured muscle strength. In this ssy, the nimls were plced on wire mesh tht ws then inverted, nd their ltency to fll recorded. As with the rotrod ssy, mesurements were mde monthly strting when the nimls were 4. months old. At this ge, ll the Pompe mice showed deficit in muscle strength when compred to wild type mice, s indicted y their decresed ltency (Figure 4c). As expected, vehicle-treted mice demonstrted continul decline in muscle strength s the disese progressed. Pompe mice dosed with either or 1 mg/kg of the unmodified enzyme (strting t. months of ge) initilly demonstrted similr decline in performnce ut ppered to stilize fter 3 months of tretment. However, these improvements did not rech sttisticl significnce until fter 7 months of tretment (Figure 4c). In contrst, the cohort of Pompe mice dministered mg/kg oxime-neo-rhgaa showed no diminution in muscle strength throughout the durtion of the study. Although they did not improve to the level oserved in wild type mice, their performnce in this ssy ws significntly greter thn tht oserved with oth the untreted nd rhgaa-treted groups (Figure 4c). This result suggests tht nimls treted with the modified enzyme responded more roustly thn those treted with the unmodified counterprt, even when dministered fivefold lower dose. Reducing lysosoml glycogen levels in older Pompe mice did not trnslte to significnt improvements in motor function To determine if the virtues shown ssocited with oxime-neorhgaa could e recpitulted in older, more symptomtic Pompe mice, similr study ws performed using mice strting t 1 months of ge (insted of. months). Ten-month-old Pompe mice hve higher levels of ccumultion of lysosoml glycogen nd demonstrte impired motor function. As with the previous study design, 1-month-old Pompe mice were first treted with AAV8/DC19-GAA D44N to induce immunotolernce to the humn enzyme; 1 month post-tretment with the virl vector, the nimls were injected weekly with 4 mg/kg rhgaa, 1 mg/kg rhgaa, 4 mg/kg oxime-neo-rhgaa, or vehicle for months. A higher (4 mg/kg) nd more frequent (weekly) dosing regimen Glycogen content (mg/g tissue) Ltency (seconds) c Ltency (seconds) Vehicle 4 mg/kg rhgaa 1 mg/kg rhgaa 4 mg/kg oxime-neo-rhgaa Hert Diphrgm Qudriceps Triceps Time of tretment (months) Figure Assessment of motor coordintion nd muscle strength fter enzyme therpy of older, symptomtic Pompe mice. Pompe mice (1 months old) were first immunotolerized to humn GAA y dministering 7E11 drp AAV8/DC19-GAA D44N nd fter 1 month they were sujected to weekly injections with vrying doses of either rhgaa or oxime-neo-rhgaa for months. () At the end of the study, the mice were killed nd their tissues nlyzed for glycogen levels. Dt re expressed s mens ± SD (n = 1 nimls per group). Throughout the study, the nimls were sujected to () rocking rotrod nd (c) wire-hng tests. Pompe mice treted with vehicle (closed circle), 4 mg/ kg rhgaa (closed tringle), 1 mg/kg rhgaa (closed dimond), nd 4 mg/kg oxime-neorhgaa (open circle) were tested monthly. drp, DNse resistnt prticle; GAA, cid α-glucosidse; rhgaa, recominnt humn cid α-glucosidse. ws used ecuse older Pompe mice re reportedly more refrctory to tretment. 2,26 Anlysis of tissue glycogen levels t the end of the study showed ner-complete correction of the storge pthology in the hert nd diphrgm of nimls treted with either form of rhgaa (Figure ). In the qudriceps nd triceps, tretment with 4 mg/kg oxime-neo-rhgaa ws s effective s tretment with 1 mg/kg of the unmodified enzyme t reducing glycogen levels (Figure ), consistent with the oservtions noted erlier. The extent of reduction in storge mteril in the skeletl muscles of treted mice ws significnt ut incomplete (~7 9% of stored glycogen ws clered with tretment). Interestingly, despite significnt reduction in glycogen levels in the skeletl muscles, mesurements of motor function using 98 vol. 17 no. 6 june 9

6 The Americn Society of Gene Therpy Second Genertion Enzyme for Pompe Disese the rotrod nd wire-hng ssys showed only mrginl improvements over the vehicle-treted controls (Figure,c). Tretment with either the modified or unmodified rhgaa resulted in some improvement over the control mice when ssyed using the rocking rotrod tht hd een djusted to spin t lower velocity (rotrod ws progrmmed to spin t speed of rpm/2. seconds insted of 17. rpm/second). However, we could not detect ny differences in performnce etween the three enzyme-treted groups (Figure ). In the wire-hng test, the performnce of the enzyme-treted mice ws not different from tht of the vehicletreted cohort (Figure c). These results indicted tht reduction in glycogen storge levels per se in older Pompe mice did not necessrily trnslte to improvements in muscle function. c d Erly therpeutic intervention with oxime-neo-rhgaa ted the extent of degenertion nd regenertion of muscle fiers in Pompe mice To exmine the sis for the oserved difference in functionl response to enzyme therpy etween nimls tht were treted erlier versus lter, histopthologicl nlysis of the muscles ws performed. In helthy muscle fiers, s expected, the nuclei were primrily locted t the periphery of the cells (Figure 6). A centronucler ppernce is suggestive of degenertive nd regenertive event s my occur fter injury or serious myopthy. Exmintion of the qudriceps from 3-month-old Pompe mice showed similr pttern of peripherl locliztion of the nuclei s oserved in wild type mice (Figure 6). However, when the Pompe nimls reched months (Figure 6c) or 1 months (Figure 6d) of ge, progressively greter numers of myofiers were oserved to hror centrlly loclized nuclei. Quntittion of the numer of muscle fiers with centronucler ppernce (collted from ~2, myofiers from different sections of three Pompe mice t ech time point) showed n increse in the numer of cells with this phenotype s function of ge, reflecting the progressive nture of the disese (Figure 7). The numer would pper to stilize fter 1 months suggesting perhps loss of regenertive cpcity. In ddition to hving lrger numer of centrlly loclized nuclei, the sections from the older Pompe mice lso displyed evidence of ccumultion of glycogen in the cells (visulized s white, punctte spots). Skeletl muscles from Pompe mice treted with enzyme strting t. months of ge nd nlyzed 8 months lter showed evidence of qulittive nd quntittive chnges. Pompe mice treted with iweekly infusions of mg/kg rhgaa showed reduction in the numer of glycogen deposits ut no difference in the numer muscle fiers with centronucler ppernce (~%) compred to those noted in untreted, ge-mtched Pompe mice (Figures 6e,f nd 7). However, those treted with 1 mg/kg rhgaa (Figure 6g) or mg/kg oxime-neo-rhgaa (Figure 6h) showed lower numer of fiers (~12%) with centrl nuclei nd greter clernce of glycogen deposits. The numer of fiers with centronucler ppernce in these nimls t the conclusion of the study period (8 months post-tretment) ws similr to tht noted t the strt of the therpy, when the nimls were. months old (Figure 7). This suggested tht erly tretment with either 1 mg/ kg of the unmodified enzyme or mg/kg of oxime-neo-rhgaa essentilly prevented further degenertion nd regenertion e g Figure 6 Histopthologicl nlysis of skeletl muscles of Pompe mice. Representtive sections of qudriceps from Pompe mice of different ges nd fter enzyme therpy were fixed in 1% neutrl uffer formlin nd stined with hemtoxylin nd eosin. The extent of glycogen ccumultion (visulized s white deposits) nd centronucletion ws noted in tissues otined from representtive tissues ( mgnifiction) of () 3-month-old wild type mouse, () 3-month-old Pompe mouse, (c) -month-old Pompe mouse nd (d) 1-month-old Pompe mouse. Pnel (e) ws sectioned from 13.-month-old Pompe mouse treted with vehicle for 8 months; (f) ws from 13.-month-old Pompe mouse tht received mg/kg rhgaa eginning t. months of ge for totl of 8 months; pnel (g) ws otined from Pompe mouse tht received 1 mg/kg rhgaa for 8 months nd pnel (h) from one tht received mg/kg oxime-neo-rhgaa for 8 months. Arrows denote the loction of myofiers with centronucletion. rhgaa, recominnt humn cid α-glucosidse. of the muscle fiers. These results were lso consistent with the iochemicl nd motor function dt showing tht mg/kg of unmodified rhgaa ws less efficcious thn 1 mg/ kg of the sme enzyme or mg/kg of oxime-neo-rhgaa. Anlysis of the skeletl muscles of Pompe mice tht received enzyme therpy strting t 1 months of ge (when the disese ws more dvnced) showed no chnge in the frequency of myofiers with centrl nuclei when compred to vehicle-treted controls (Figure 7). Irrespective of whether they were dministered vehicle, unmodified or modified enzyme, the numer of cells with f h Moleculr Therpy vol. 17 no. 6 june 9 99

7 Second Genertion Enzyme for Pompe Disese The Americn Society of Gene Therpy % Of fiers with centrlized nuclei % Of fiers with centrlized nuclei Vehicle mg/kg rhgaa 1 mg/kg rhgaa mg/kg oxime-neo-rhgaa Age of mice (months) Vehicle 4 mg/kg rhgaa 1 mg/kg rhgaa 4 mg/kg oxime-neo-rhgaa Age of mice (months) Figure 7 Extent of degenertion nd regenertion in Pompe mouse muscles treted t different ges. Hemtoxylin nd eosin stined sections from three nimls of ech tretment group (t ech time point) ws scored for the numer of myofiers hroring centrlized nuclei. For ech niml, 1, 3, muscle fiers from different fields were exmined. The results illustrted in were from Pompe mice tht were treted with iweekly infusions of enzyme strting t. months of ge, nd those in () were from Pompe mice treted weekly with enzyme infusions strting when they were 11 months old. Dt re presented s mens ± SD. Dshed lines represent the time periods the nimls were treted. centrl nuclei ws the sme in ll the Pompe nimls when their muscles were nlyzed t months post-tretment (Figure 7). The high numer of myofiers with the centronucler phenotype (~%) suggests tht delyed therpeutic intervention ws not effective t reversing muscle dmge. Exposure of the muscles to chronic insult from sustined ccumultion of glycogen in the lysosomes ws likely responsile for the development of the oserved muscle pthology. Reducing the urden of glycogen ccumultion in these more ffected muscles ws not sufficient to improve motor function, t lest when tested fter months of enzyme therpy. Discussion The experience with enzyme replcement therpy for Pompe disese suggests tht more efficient delivery of the recominnt enzyme, prticulrly to the skeletl muscle, my improve clinicl response. Fctors tht my e contriuting to this refrctoriness include the reltively poor ccessiility of the skeletl muscle to infused rhgaa, the lrge mss of this orgn, the purported differentil response of different muscle fier types to therpy, nd the low levels of the CI-MPR tht is responsile for intrcellulr uptke of the enzyme in this tissue. 16,27,28 To ddress this chllenge, rhgaa ws glycoengineered to enhnce its recognition y the cell surfce receptor nd therey increse its uptke y the ffected tissues. Mnipulting the crohydrte moieties of glycoproteins s n pproch to modulte their properties hs een used successfully for vriety of therpeutics. The growing doption of this strtegy is orne of the recognition tht glycosyltion impcts numer of functions, including protein s phrmcokinetic nd phrmcodynmic profile, s well s its soluility, stility, intrcellulr trfficking, nd receptor inding ctivity For exmple, hyperglycosyltion of erythropoietin nd follicle stimulting hormone through the introduction of dditionl sites for N-linked glycosyltion incresed their serum hlf-lives nd enhnced their iologicl ctivities Engineered cell lines tht produce fucosylted monoclonl ntiody therpeutics incresed their ntiody- dependent cell cytotoxic ctivities. 3 These exmples employed strtegies tht either ltered the coding sequence of the protein such tht they encoded for dditionl sites of glycosyltion or involved re-engineering the mchinery of glycosyltion in expression systems to produce more ctive iologic. Another pproch to lter the crohydrte moieties on therpeutic proteins is to effect wht is sometimes referred to s synthetic posttrnsltionl modifiction. This modifiction is usully imposed fter protein production is completed nd frequently during downstrem processing of the protein. The remodeling of the oligoscchrides on recominnt glucocererosidse to generte Cerezyme 4 nd the cyltion of insulin to generte Levemir 36 re exmples of this pproch. Yet nother illustrtion of synthetic post-trnsltionl modifiction is the chemicl or enzymtic conjugtion of polyethylene glycol onto therpeutic proteins to reduce their immunogenicity or decrese their rte of clernce from the lood. Polyethylene glycolted iophrmceuticls tht hve gined regultory pprovl over the pst few yers include polyethylene glycolted interferon (Pegsys) nd humn growth hormone (Somvert). 37,38 Our effort to lter the rchitecture of the oligoscchrides on GAA to present higher numers of M6P residues represents novel ppliction of this concept. We hve evluted two pproches to decorte the oligoscchrides on GAA with terminl M6P residues. One involves the stepwise enzymtic modifiction of rhgaa using N-cetylglucosmine-1- phosphotrnsferse nd N-cetylglucosmine-1-phosphodiester α-n-cetylglucosminidse to generte wht is referred to s HP-GAA. 19,39,4 However, testing of this hyper-mnnose 6-phosphorylted vrint showed tht it ws no more effective thn unmodified enzyme t clering tissue glycogens from Pompe mice. This result ws due to HP-GAA presenting dditionl mnnose residues tht led to the nonproductive sequestrtion of the infused enzyme y mnnose receptors on endothelil cells nd mcrophges. 19 To overcome this impediment, we developed n lternte pproch tht involved chemiclly conjugting synthetic oligoscchride ering M6P residues directly onto rhgaa to generte oxime-neo-rhgaa. The cronyl-coupled oxime chemistry employed for conjugtion could e ccomplished under conditions tht did not significntly ffect the enzyme s ctivity. The rection ws lso efficient nd reproducile nd the resultnt product ws stle nd conducive to mnufcturing t scle vol. 17 no. 6 june 9

8 The Americn Society of Gene Therpy Second Genertion Enzyme for Pompe Disese In this regrd, rhgaa contining the oxime ond represented n improvement over tht contining the hydrzone ond reported previously. 21 The resulting modified rhgaa (oxime-neo-rhgaa) displyed higher ffinity for the CI-MPR with consequent greter receptor-medited endocytosis into L6 myolst cells in vitro nd presumly into Pompe-ffected muscles in vivo. This trnslted to n pproximtely fivefold higher efficcy in clering the tissue glycogen, sed on the ility of the oxime-neo-rhgaa to effect n equivlent reduction in sustrte levels t one-fifth the dose of the unmodified enzyme. Importntly, this improved clernce of glycogen resulted in corresponding increse in muscle strength nd motor function in mice treted strting t 6 months of ge. The oserved improvement in motor function ws not due to reduction of glycogen in the CNS of Pompe mice. 41 Although mnnose 6-phosphorylted β-glucuronidse ws reportedly le to trverse the lood rin rrier of mucopolyscchridosis VII mice, 42 we were not le to detect either GAA ctivity or reduction in glycogen in the CNS of oxime-neo-rhgaa-treted Pompe mice (dt not shown). Surprisingly, lthough comprle reductions in glycogen levels were oserved in Pompe mice treted with mg/kg oximeneo-rhgaa nd 1 mg/kg rhgaa, the improvement in muscle function ws greter in nimls dministered the crohydrteengineered enzyme. It my e tht oxime-neo-rhgaa effected more rpid clernce of glycogen nd therey reduced the time the muscles were exposed to the insult. This in turn my hve reduced or minimized the muscle dmge of oxime-neo-rhgaa-treted mice compred to those dministered rhgaa. Preventing the onset of muscle dmge is n importnt considertion, s tretment of older Pompe mice with more estlished muscle pthology ws lrgely ineffective t rescuing motor function. Clernce of glycogen from these older nd more dmged muscles ws insufficient to improve their performnce on the rotrod or wirehng tests. This suggests tht other fctors esides glycogen hve role in modulting muscle function. The importnce of erly intervention hs lso een noted in humn clinicl studies, in which more roust outcome ws oserved in infntile Pompe sujects treted efore they were 6 months old thn in those treted etween 6 nd 36 months of ge. 13,43 The sis for this differentil response in nimls treted erly nd lte is uncler ut my e relted to the reported decresed ility of older Pompe mice muscles to tke up the enzyme. 21,2,26 More dmged muscle fiers, prticulrly type II fiers, lso reportedly exhiit ltered utophgic ctivity tht my ffect norml trfficking of the endocytosed enzyme. 44 Of dditionl relevnce ws the oservtion tht mice treted erlier hd fewer numers of fiers with centrlized nuclei, mrker of muscle degenertion nd regenertion. Although oth erly nd lte therpeutic interventions prevented further increses in the numers of fiers contining centrlized nuclei, the sl numers in the older nimls were much higher. Hence, the inility to rescue muscle function in the older mice my hve een due to loss in their cpcity to repir dmged fiers, perhps due to exhustion of stellite cells. This loss of regenertive cpcity hs een reported for other degenertive muscle diseses nd severe firotic diseses, the ltter due to n inility of stellite cells to fuse with the dmged muscle fiers. 4 Irrespective of the mechnism, the results here strongly dvocte the desire for erly dignosis nd therpeutic intervention in the mngement of Pompe disese. Another considertion is the use of djuvnt therpies designed to improve muscle physiology such s y codministering insulinlike growth fctor-1 nd ntgonists of myosttin. Both gents hve reportedly shown promise in improving muscle function in mouse models of Duchenne musculr dystrophy. 46,47 In summry, we showed tht the pproch of chemiclly conjugting lignd onto rhgaa to enhnce its recognition y the CI-MPR significntly improved the enzyme s potency t clering tissue glycogen levels from Pompe mice. Importntly, this trnslted to concomitnt dely in disese progression nd n increse in motor function. The vilility of this improved enzyme (oxime-neo-rhgaa) mens tht either lower dose of this enzyme could e used for tretment or tht dose comprle with tht of the unmodified enzyme could provide etter efficcy. The use of lower doses could lessen the mgnitude of the host immune response to the dministered therpeutic which in turn could positively impct the efficcy of tretment. The cronyl chemistry used for conjugtion ws reltively innocuous to the enzyme s ctivity nd ws conducive to scle up. Bsed on these oservtions, we contend tht this pproch to glycoengineering could e rodly pplied to other therpeutics (esides lysosoml enzymes) ering different synthetic lignds designed to trget delivery vi their respective cognte receptors to disese tissues. As such, this represents generl pproch for optimizing therpeutic protein leds nd thus cn e pplied to developing new nd improved protein phrmceuticls. Mterils nd Methods Chemicls. Sodium met-periodte, M6P, β-glycerophosphte, 4- methylumelliferyl-α-d-glucopyrnoside, Aspergillus niger myloglucosidse, ovine liver glycogen, nd other common chemicl regents were purchsed from Sigm Chemicl (St. Louis, MO) unless otherwise stted. N-(t-BOC)-minooxycetic cid tetrfluorophenyl ester, fetl ovine serum, Dulecco s modified Egle s medi nd other cell culture regents were from Invitrogen (Crlsd, CA). Recominnt humn rhgaa purified from Chinese hmster ovry cells ws from Genzyme (Cmridge, MA). Dilysis cssettes of vrying cpcities nd with moleculr weight cutoff of 1 kd were from Pierce (Rockford, IL). Difiltrtion crtridges were otined from GE Amershm Biosciences (Pisctwy, NJ). The Amplex Red glucose ssy kit ws from Moleculr Proes (Eugene, OR). Modifiction of the glycn to contin n minooxy group. The originl synthetic oligoscchride ws derivtized with utyl hydrzide for coupling to rhgaa through hydrzone ond. 21 To convert the hydrzide group to n minooxy group for oxime ond formtion, N-(t-BOC)- minooxycetic cid tetrfluorophenyl ester (t-boc-aoaa-tfp) ws used. The synthetic glycn nd t-boc-aoaa-tfp were dissolved seprtely in DMSO nd wter (:, vol:vol) to finl concentrtion of.3 mmol/l nd mmol/l, respectively. Then two volumes of the synthetic glycn were mixed with 1 volume of t-boc-aoaa-tfp such tht the finl molr rtio of glycn:t-boc-aoaa-tfp ws 1:3.7. Following mixing, 3-hydroxy- 1,2,3-enzotrizin-4(3H)-one (Dht-OH) from stock (196.6 mmol/l) ws dded such tht the glycn:t-boc-aoaa-tfp:dht-oh rtio ws 1:3.7:1.8. The rection ws llowed to proceed overnight t room temperture with gentle shking. Completion of the rection ws verified y Dionex nlysis. The glycn-minooxy-t-boc in the rection mixture ws purified y gel filtrtion over Sephdex G-1 column (Sigm Chemicl) Moleculr Therpy vol. 17 no. 6 june 9 961

9 Second Genertion Enzyme for Pompe Disese The Americn Society of Gene Therpy using distilled H 2 O s eluent; the frctions contining the rection product were collected nd lyophilized. Deprotection of the lyophilized glycnminooxy-t-boc ws performed in % trifluorocetic cid in dichlorormethne t room temperture for 3 minutes fter which the solvent ws removed under nitrogen. The finl mteril ws dissolved in. mol/l sodium cette uffer, ph 3 4, nd frctionted on Sephdex G-1 column s descrie ove. Frctions contining the finl product were collected nd lyophilized. Overll yield ws >9% of the strting mteril. Conjugtion of synthetic glycn onto rhgaa. The procedure for chemiclly conjugting the derivtized synthetic oligoscchride onto rhgaa ws essentilly s descried previously. 21 Briefly, mg to 1 g of periodteoxidized rhgaa were conjugted to the minooxy-derivtized synthetic oligoscchride y mixing t molr rtio of 1:1 nd incuting t 37 C for 6 hours with gentle shking. Following conjugtion, the preprtions were difiltrted using dilfiltrtion crtridge (moleculr weight cutoff of 3 kd) with volumes of 2 mmol/l sodium phosphte uffer, ph 6.2. Following difiltrtion, the oxime-neo-rhgaa ws djusted to contin 2% mnnitol nd.% Tween-8 nd then sterile filtered. The smples were liquoted, snp-frozen on dry ice nd stored t 8 C until used. The numer of M6P residues on rhgaa nd oxime-neo-rhgaa ws quntitted using the method descried y Zhou et l. 48 Chrcteriztion of oxime-neo-rhgaa. Methods to mesure the inding of oxime-neo-rhgaa to CI-MPR column nd to ssy the uptke of the enzyme into L6 myolst cells hve een descried previously.,21 To mesure enzyme ctivity, cells were first lysed in ssy uffer (.2 mol/l sodium cette,.4 mol/l potssium chloride, ph 4.3) contining.1% Triton X-1. Following centrifugtion to remove cellulr deris, the superntnts were ssyed for enzymtic ctivity using the fluorogenic sustrte 4-methylumelliferyl-α-d-glucopyrnoside. In studies to determine the stility of the conjugtes, the different preprtions were incuted t 4 C or room temperture (2 C) for vrious times. Smples were snp-frozen on dry ice nd stored t 8 C until ll the time points were collected. They were then trnsferred into DiLyzer (Pierce) with moleculr weight cutoff of 3 kd nd dilyzed overnight ginst 2 mmol/l phosphte uffer, ph 6.2 t 4 C. Smples were collected nd their M6P content then mesured. Genertion of AAV8/DC19-GAAD44N. The previrl plsmid psp7/ DC19-GAA contining the cdna for humn GAA nd liver-specific promoter (DC19) ws generted s descried in Ziegler et l. 2 AAV8/ DC19-GAA D44N encodes humn GAA hroring muttion (D44N) tht is locted in the ctive site. 22 The terminology used is modified such tht the D18N muttion referred to in the originl puliction is D44N s descried herein. The expression cssette flnked y AAV2 ITRs ws pckged into AAV8 cpsids using stndrd triple trnsfection protocol. AAV8 pseudotyped vectors were purified y iodixnol grdient centrifugtion followed y ion exchnge chromtogrphy over Hi Trp Q HP columns. The concentrtion of DNse resistnt prticles ws determined using reltime TqMn PCR ssy (ABI PRISM 77; Applied Biosystems, Foster City, CA) with primers designed to mplify the vector-specific ovine growth hormone polydenyltion sequence. Short-term efficcy studies. Animl experiments were conducted in ccordnce with the guidelines issued y the US Deprtment of Helth nd Humn Services (NIH Puliction No ) nd y Genzyme s IACUC committee. The mouse model of Pompe disese (6 neo /6 neo ) 2,28 tht hd een crossed onto predominntly C7BL/6J genetic ckground recpitultes spects of the humn disese in tht they hror mutted GAA gene, exhiit no mesurle α-glucosidse ctivity, nd lysosoml ccumultion of glycogen in muscle. The short-term efficcy studies used young (~ 6 months old) Pompe mice. Groups of Pompe mice (n = 8) were injected vi til vein with vehicle or vrying doses of rhgaa or oxime-neo-rhgaa. The mice were dministered 4 weekly doses nd then scrificed 2 weeks fter the lst tretment. To minimize the hypersensitivity rections frequently oserved fter repeted dministrtions of rhgaa, mice were routinely pretreted with mg/kg diphenhydrmine (delivered intrperitonelly) 1 minutes efore injection of the enzyme preprtions. Vrious tissues including the hert, diphrgm, nd skeletl muscles (qudriceps nd triceps) were collected nd stored t 8 C until ssyed. Long-term efficcy studies. Longer-term studies were performed with either young (~4. months of ge, n = 1) or older (~1 months of ge, n = 1) Pompe mice. For these studies, nimls were first immunotolerized y dministering E11 (young mice) or 7E11 (older mice) DNse resistnt prticle of AAV8/DC19-GAA D44N vi til vein. Strting 1 month lter, nimls were sujected to periodic injections with different enzyme preprtions. Motor function ws ssessed using vriety of tests. A rotrod test in the rocking mode ws used to exmine motor function. 2 The speed of the rotrod used to test the young Pompe mice ws set to increse to 17. rpm within 2. seconds nd the direction of spin then reversed to ccelerte in the opposite direction. For the older Pompe mice, the speed ws set to increse to rpm within 2. seconds. Time ws recorded s the ltency of the niml to sty on the pprtus. Wire-hng tests were used to mesure muscle strength. Individul nimls were plced on metl mesh grid tht ws then inverted; the time the niml ws le to hold on the mesh in n up-side-down position ws recorded. For ech time point, three tests were typiclly performed for ech niml nd the verge reding recorded. Mesurement of glycogen levels. The glycogen content in vrious muscles of the Pompe mice ws ssyed y mesuring the difference in the mount of glucose relesed from oiled tissue homogente following digestion with or without A. niger myloglucosidse, s descried previously.,21 The glucose levels from the digested nd undigested smple sets were then ssyed using the Amplex Red glucose ssy kit ccording to the mnufcturer s instructions. Bovine liver glycogen ws used s stndrd. Histopthology. Qudriceps from Pompe mice ws fixed in 1% formlin nd emedded in prffin. They were then sectioned to -μm thickness nd stined with hemtoxylin nd eosin for ssessment of centrlized nuclei. Tissue sections from three nimls in ech group were scored for the numer of muscle fiers hroring centrlized nuclei. On verge, totl of 1, 3, muscle fiers from different fields were exmined for ech niml. For periodic cid-schiff stining, tissues were fixed in 3% glutrldehyde, emedded in epon nd then stined with periodic cid-schiff. Sttisticl nlysis. Behviorl dt re expressed s men ± stndrd devition. Dt were nlyzed y one-wy nlysis of vrince followed y Bonferroni s multiple comprison tests with GrphPd Prism 4 softwre (GrphPd Softwre, L Joll, CA). A proility vlue of P <. ws considered sttisticlly significnt. Acknowledgments We thnk the scientific community t Genzyme for their vlule discussions nd input into the designs of the studies. We re especilly thnkful to memers of the Deprtments of Comprtive Medicine, Bioengineering, Applied Discovery Reserch, Histology, Protein Therpy Reserch, Glycoiology, nd Therpeutic Protein Development for providing regents nd technicl support. We thnk Ron Scheule for his help with editing nd proof-reding the mnuscript. We re very grteful to Nin Ren t NIH for providing the Pompe mice. Finlly, we would like to thnk Jonthn Fidler nd Michel Zho for their help with muscle function tests, nd Qun Zhou nd Josephine Kyzike for M6P nlysis. References 1. Koury, MJ (3). Sugr coting extends hlf-lives nd improves effectiveness of cytokine hormones. Trends Biotech 21: Elliott, S, Lorenzini, T, Asher, S, Aoki, K, Brnkow, D, Buck, L et l. (3). Enhncement of therpeutic protein in vivo ctivities through glycoengineering. Nt Biotechnol 21: vol. 17 no. 6 june 9

10 The Americn Society of Gene Therpy Second Genertion Enzyme for Pompe Disese 3. Lu, D, Kuzm, G, Wei, CM, Livingston, DJ nd Hsiung, N (1987). A modified humn tissue plsminogen ctivtor with extended hlf-life in vivo. Nt Biotechnol : Furish, FS, Steer, CJ, Krett, NL nd Brrnger, JA (1981). Uptke nd iodistriution of plcentl glucocererosidse in rt heptic cells nd effects of sequentil deglycosyltion. Biochim Biophys Act 673: Brdy, RO, Murry, GJ nd Brton, NW (1994). Modifying exogenous glucocererosidse for effective replcement therpy in Gucher disese. J Inherit Met Dis 17: Hirschhorn, R nd Reuser, AJJ (1). Glycogen storge disese type II: cid α-glucosidse (cid mltse) deficiency. In: CR Scriver, AL Beudet, WS Sly nd D Vlle (eds). The Metolic nd Moleculr Bsis of Inherited Disese. McGrw-Hill: New York. pp Vn der Ploeg, AT nd Reuser, AJJ (8). Pompe s disese. Lncet 372: Vn den Hout, HM, Hop, W, vn Diggelen, OP, Smeitink, JA, Smit, GP, Poll-The, BT et l. (3). The nturl course of infntile Pompe s disese: originl cses compred with 133 cses from the literture. Peditrics 112: Kishnni, PS nd Howell, RR (4). Pompe disese in infnts nd children. J Peditr 144: S3 S Felice, KJ, Alessi, AG nd Grunnet, ML (199). Clinicl vriility in dult-onset cid mltse deficiency; report of ffected sis nd review of the literture. Medicine 74: Vorgerd, M, Burwinkel, B, Reichmnn, H, Mlin, JP nd Kilimnn, MW (1998). Adult-onset glycogen storge disese type II: phenotypic nd llelic heterogeneity in Germn ptients. Neurogenetics 1: Kroos, MA, Pomponio, RJ, Hgemns, ML, Keulemns, JL, Phipps, M, DeRiso, M et l. (7). Brod spectrum of Pompe disese in ptients with the sme c t G hplotype. Neurology 68: Kishnni, PS, Nicolino, M, Voit, T, Rogers, RC, Tsi, AC, Wterson, J et l. (6). Chinese hmster ovry cell-derived recominnt humn cid α-glucosidse in infntile-onset Pompe disese. J Peditr 149: Rossi, M, Prenti, G, Dell Cs, R, Romno, A, Mnsi, G, Agovino, T et l. (7). Long-term enzyme replcement therpy for Pompe disese with recominnt humn α-glucosidse derived from Chinese hmster overy cells. J Child Neurol 22: Vn Cpelle, CI, Winkel, LP, Hgemns, ML, Shpir, SK, Arts, WF, vn Doorn, PA et l. (8). Eight yers experience with enzyme replcement therpy in two children nd one dult with Pompe disese. Neuromuscul Disord 18: Wenk, J, Hille, A nd von Figur, K (1991). Quntittion of Mr 46 nd Mr 3 mnnose 6-phospht receptors in humn cells nd tissues. Biochem Int 23: Funk, B, Kessler, U, Eisenmenger, W, Hnsmnn, A, Kol, HJ nd Kiess, W (1992). Expression of the insulin-like growth fctor-ii/mnnose 6-phosphte receptor in multiple humn tissues during fetl life nd erly infncy. J Clin.Endocrinol Met 7: Ghosh, P, Dhms, S nd Kornfeld, S (3). Mnnose 6-phosphte receptors: new twists in the tle. Nt Rev 4: McVie-Wylie, AJ, Lee, KL, Qiu, H, Jin, X, Do, H, Gotschll, R et l. (8). Biochemicl nd phrmcologicl chrcteriztion of different recominnt cid α-glucosidse preprtions evluted for the tretment of Pompe disese. Mol Genet Met 94: Zhu, Y, Li, X, Kyzike, J, Zhou, Q, Thurerg, BL, Ren, N et l. (4). Conjugtion of mnnose 6-phosphte-contining oligoscchrides to cid α-glucosidse improves the clernce of glycogen in Pompe mice. J Biol Chem 279: Zhu, Y, Li, X, McVie-Wylie, AJ, Jing, C, Thurerg, BL, Ren, N et l. (). Crohydrte-remodelled cid α-glucosidse with higher ffinity for the ctionindependent mnnose 6-phosphte receptor demonstrtes improved delivery to muscles of Pompe mice. Biochem J 389: Hermns, MMP, Kroos, MA, vn Beeumen, J, Oostr, BA nd Reuser, AJJ (1991). Humn lysosoml α-glucosidse. Chrcteriztion of the ctlytic site. J Biol Chem 266: Mingozzi, F, Liu, YL, Dorzynski, E, Kufhold, A, Liu, JH, Wng, Y et l. (3). Induction of immune tolernce to cogultion fctor IX y in vivo heptic gene trnsfer. J Clin Invest 111: Ziegler, RJ, Cherry, M, Bron, CM, Li, C, Bercury, SD, Armentno, D et l. (7). Correction of the iochemicl nd functionl deficits in Fry mice following AAV8- medited heptic expression of α-glctosidse A. Mol Ther 1: Ziegler, RJ, Bercury, SD, Fidler, J, Zho, MA, Foley, J, Tksir, T et l. (8). Aility of AAV8-medited heptic expression of cid α-glucosidse to correct the iochemicl nd motor function deficits of presymptomtic nd symptomtic Pompe mice. Hum Gene Ther 6: Ren, N, Fukud, T, Gilert, AL, de Jong, D, Thurerg, BL, Mttlino, RJ et l. (). Replcing α-glucosidse in Pompe disese: recominnt nd trnsgenic enzymes re equipotent, ut neither completely clers glycogen from type II muscle fiers. Mol Ther 11: Thurerg, BL, Lynch Mloney, C, Vccro, C, Afonso, K, Tsi, AC, Bossen, E et l. (6). Chrcteriztion of pre- nd post-tretment pthology fter enzyme replcement therpy for Pompe disese. L Invest 86: Ren, N, Jtkr, T, Lee, A, Lu, N, Dwivedi, S, Ngrju, K et l. (2). Glycogen stored in skeletl ut not in crdic muscle in cid α-glucosidse mutnt (Pompe) mice is highly resistnt to trnsgene-encoded humn enzyme. Mol Ther 6: Wlsh, G nd Jefferis, R (6). Post-trnsltionl modifictions in the context of therpeutic proteins. Nt Biotechnol 24: Cmpell, CT, Smpthkumr, S nd Yrem, KJ (7). Metolic oligoscchride engineering: perspectives, pplictions, nd future directions. Mol Biosyst 3: Werner, RG, Kopp, K nd Schlueter, M (7). Glycosyltion of therpeutic proteins in different production systems. Act Peditr 96: Egrie, JC, Dwyer, E, Browne, JK, Hitz, A nd Lykos MA (3). Drepoeitin lf hs longer circulting hlf-life nd greter in vivo potency thn recominnt humn erythropoietin. Exp Hemtol 31: Perlmn, S, vn den Hzel, B, Christinsen, J, Grm-Nielsen, S, Jeppesen, CB, Andersen, KV et l. (3). Glycosyltion of n N-terminl extension prolongs the hlf-life nd incresed the in vivo ctivity of follicle stimulting hormone. J Clin Endocrinol Met 88: Sinclir, AM nd Elliott, S (). Glycoengineering: the effect of glycosyltion on the properties of therpeutic proteins. J Phrm Sci 94: Ymne-Ohnuki, N, Kinoshit, S, Inoue-Urkuo, M, Kusunoki, M, Iid, S, Nkno, R et l. (4). Estlishment of FUT8 knockout Chinese hmster ovry cells: n idel host cell line for producing completely defucosylted ntiodies with enhnced ntiody-dependent cellulr cytotoxicity. Biotechnol Bioeng 87: Goldmn-Levine, JD nd Lee, KW (). Insulin detemir new sl insulin nlog. Ann Phrmcother 39: Prkinson, C nd Triner, PJ (1). The plce of pegvisomnt in the mngement of cromegly. Expert Opin Investig Drugs 1: Foster, GR (4). Pegylted interferons: chemicl nd clinicl differences. Ailment Phrmcol Ther : Kornfeld, R, Bo, M, Brewer, K, Noll, C nd Cnfield, WM (1998). Purifiction nd multimeric structure of ovine N-cetylglucosmine-1-phosphodiester α-ncetylglucosminidse. J Biol Chem 273: Kudo, M nd Cnfield, WM (6). Structurl requirements for efficient processing nd ctivtion of recominnt humn UDP-N-cetylglucosmine:lysosoml-enzyme- N-cetylglucosmine-1-phosphotrnsferse. J Biol Chem 281: Sidmn, RL, Tksir, T, Fidler, J, Zho, M, Dodge, JC, Pssini, MA et l. (8). Temporl neuropthologic nd ehviorl phenotype of 6neo/6neo Pompe disese mice. J Neuropthol Exp Neurol 67: Urym, A, Gru, JH, Sly, WS nd Bnks, WA (4). Developmentlly regulted mnnose 6-phosphte receptor-medited trnsport of lysosoml enzyme cross the lood-rin rrier. Proc Ntl Acd Sci USA 11: Kishnni, PS, Corzo, D, Nicolino, M, Byrne, B, Mndl, H, Hwu, WL et l. (7). Recominnt humn α-glucosidse: mjor clinicl enefits in infntile-onset Pompe disese. Neurology 68: Fukud, T, Ahern, M, Roerts, A, Mttlino, RJ, Zl, K, Plotz, PH et l. (6). Autophgy nd mistrgeting of therpeutic enzyme in skeletl muscle in Pompe disese. Mol Ther 14: Jejurik, SS nd Kuzon, WM (3). Stellite cell depletion in degenertive skeletl muscle. Apoptosis 8: Ptel, K, Mchri, R nd Amthor, H (). Moleculr mechnisms involving IGF-1 nd myosttin to induce hypertrophy s therpeutic strtegy for Duchenne musculr dystrophy. Act Myol 24: Nktni, M, Tkehr, Y, Sugino, H, Mtsumoto, M, Hshimoto, O, Hsegw, Y et l. (8). Trnsgenic expression of myosttin inhiitor derived from follisttin increses skeletl muscle mss nd meliortes dystrophic pthology in mdx mice. FASEB J 22: Zhou, Q, Kyzike, J, Edmunds, T nd Higgins, E (2). Mnnose 6-phosphte quntittion in glycoproteins using high-ph nion-exchnge chromtogrphy with pulsed mperometric detection. Anl Biochem 36: Moleculr Therpy vol. 17 no. 6 june 9 963