Vaccinia virus as a subhelper for AAV replication and packaging

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1 Cittion: Moleulr Therpy Methods & Clinil Developent (2015) 2, 150; doi: /t All rights reserved /15 Artile Vini virus s suhelper for AAV replition nd pkging Andre R Moore 1, Bio Dong 1, Lingxi Chen 1 nd Weidong Xio 1 eno-ssoited virus (AAV) hs een widely used s gene therpy vetor to tret vriety of disorders. While these vetors re inresingly populr nd suessful in the lini, there is still uh to lern out the viruses. Understnding the iology of these viruses is essentil in engineering etter vetors nd generting vetors ore effiiently for lrge-sle use. AAV requires helper for prodution nd replition king this spet of the virl life yle ruil. Vini virus () hs een widely ited s helper virus for AAV. However, to dte, there re no detiled nlyses of its helper funtion. Here, the helper role of ws studied in detil. In ontrst to oon elief, we deonstrted tht ws not suffiient helper virus for AAV replition. Vini filed to produe raav nd tivte AAV prooters. While this virus ould not support raav prodution, Vini ould initite AAV replition nd pkging when AAV prooter tivtion is not neessry. This tivity is due to the ility of Vini-driven to trnsrie in the ytopls nd susequently trnslte in the nuleus nd undergo typil funtions in the AAV life yle. As suh, is suhelper for AAV opred to oplete helper funtions of denovirus. Moleulr Therpy Methods & Clinil Developent (2015) 2, 150; doi: /t.2015.; pulished online 18 Noveer 2015 INTRODUCTION eno-ssoited virus (AAV) is replition-defiient prvovirus tht requires helper virus to oplete its lyti infetion. It ontins single genoe of pproxitely.7 k flnked y two inverted terinl repets (ITRs). The AAV genoe ontins two open reding fres, whih enodes four Rep nd three Cp proteins. 1 Helper viruses, suh s denovirus, re responsile for vrious funtions in AAV replition. enovirus ids in ny stges of the AAV life yle inluding trnsriptionl tivtion, RNA trnsport, stility nd trnsltion, 2 DNA replition nd virl ssely, nd pkging. 3,5,6 Along with denovirus, vrious other viruses hve een proposed s helper viruses for AAV replition. Herpes virus, 7 Hun Ppillo Virus, 8 nd vini virus (), 9 hve ll een reported s eing helper viruses for AAV. While the roles of Herpes virus, HPV, nd denovirus hve een investigted extensively to dte, there is only one single reserh rtile tht desries funtioning s n AAV helper virus. 9 Here, we elorte on previous findings tht ts s helper for AAV replition. Our studies found tht vini is suprie helper for AAV. Unlike typil helper viruses, suh s enovirus, vini is unle to tivte AAV prooters. Although the iportnt initil helper funtions required for AAV replition re not supplied y vini, the virus is ple of repliting the AAV genoe in the presene of nd lso funtions to ssist in pkging of AAV. Understnding the funtions of vini nd its role s helper for AAV hs ided in its usge in prodution of AAV vetors. This hs llowed for the developent of sled-up ethod for vetor prodution tht n ssist in the neessity for lrge-sle vetor nufturing required for linil use of AAV s gene therpy vetor. RESULTS filed to support raav prodution In order to evlute the previous findings tht is helper for AAV replition, we utilized the virus -T7 in vetor prodution syste to rete n AAV2-EGFP vetor. We utilized the plsid p/aav, 10 whih ontins the entire AAV2 genoe with its ntive prooters (p5, p19, nd P0) ut lking AAV ITR long with the vetor plsid psaav-cb-egfp. 11 The AAV2 vetors were produed y trnsfeting HeL ells with the two plsids nd oinfetion with -T7, denovirus s positive ontrol or ok trnsfetion. The resulting ell lyste ws evluted y susequent trnsdution of 16,095 ells nd oservtion of vetor prodution y the presene of enhned green fluoresent protein (EGFP). Figure 1 shows raav2 prtiles generted using EGFP expression s n inditor. When using denovirus s helper virus for AAV replition, infetious raav2-egfp ould e oserved. In ontrst, super-infetion filed to produe detetle ounts of raav vetor. Sine there re vrious ftors tht ould use the filure of raav prodution, the protein expression profile nd the replition of vetor DNA oth were evluted in vini-infeted, denovirus-infeted, nd ok-infeted ultures. We nlyzed the Rep nd Cp proteins expression profile using western lot nlysis. As expeted, Rep nd Cp proteins (Figure 1) were oserved in sples tht utilized denovirus s helper. However, Rep nd Cp ould not e deteted in sples tht used s helper or ok infeted sples. The replition of vetor DNA ws oserved y Southern lot nlysis of HeL ultures. Figure 1 shows tht AAV genoe replition is oserved only in the sples tht were infeted with denovirus were ultiple replition fors were 1 Deprtent of Miroiology nd Iunology, Sol Sherry Throosis Reserh Center, Teple University, Phildelphi, Pennsylvni, USA. Correspondene: W Xio (wxio@teple.edu) Reeived August 2015; epted 5 Otoer 2015

2 Helper funtion of vini virus in the AAV life yle 2 tivity. Figure 2 shows tht the AAV p5 prooter, p19 prooter, nd the p0 prooter were tivted in Hel ells in the presene of denovirus s noted y the signifint inrese in firefly luiferse tivity opred to the ok infeted ontrol. However, there is no signifint inrese in ll three AAV prooters when is dded s opred to the ok infetion ontrol. Siilr phenoen were oserved in CV-1 ells (Figure 2). The results suggested tht unlike denovirus, lone nnot tivte the AAV prooters. Rep68 Rep0 VP3 Figure 1 Vini virus lone does not support reoinnt denossoited virus (raav) prodution. HeL ells were trnsfeted with p/aav nd psaav-cb-egfp. Upon trnsfetion, ells were infeted with denovirus () or vini virus (), or ok infetion (ok) nd were hrvested t 8 hours post-infetion. () Sples were sujeted to three rounds of freezing nd thwing nd het intivted efore eing used to trnsdue 16,095 ells. raav prodution ws visulized y oservtion of EGFP expression. () Cell lyste ws prepred fro olleted ells nd protein ws extrted ording to stndrd proedure nd proed for Rep nd Cp protein. The Rep nd Cp expression profile of sples tht were infeted with denovirus or vini virus ws oserved. () For Southern lotting, 10 μg of DNA ws pplied to eh lne. Southern lotting ws perfored ording to stndrd proedures for DNA trnsfer nd hyridized with 32P-leled GFP proe. The nuer on the left indites the rker positions for 1,, nd 8 k nd the () indites the position of the onoer. oserved. Altogether, the positive ontrol (denovirus)-infeted sples showed prodution of raav2-egfp rked y EGFP expression fro superinfetion of HeL ell lyste s well Rep nd Cp protein expression nd vetor DNA replition. Viniinfeted sples filed to replite vetor DNA or produe Rep nd Cp protein, whih resulted in the lk of reoinnt AAV produed fro this ethod. This oservtion nd disrepny fro previous findings propted further investigtion into the role of vini in the AAV life yle. nnot tivte AAV prooters Sine Rep nd Cp proteins were not expressed in the ove experient, we suspeted tht the in defet of ould e due to its inility to tivte AAV prooters. In order to investigte this hypothesis, three plsids were onstruted utilizing firefly luiferse s reporter of prooter tivtion. The onstruts were designed y loning the AAV prooters into kone plsid where the prooter would drive the trnsription of firefly luiferse (Figure 2). Eh onstrut ws trnsfeted into HeL ells nd then infeted with denovirus,, or ok infetion. The ells were olleted t 8 hours post-trnsfetion nd lysed in order to detet firefly luiferse Moleulr Therpy Methods & Clinil Developent (2015) n replite the genoe in the presene of While our findings showed tht in inple of supporting AAV replition in wild-type setting, we wnted to further investigte the findings tht Vini is n AAV helper. While the previous dt shows tht Vini does not tivte the AAV p5, p19, nd p0 prooters, it y e iportnt in other spets of the AAV life yle. To investigte if y support AAV replition when AAV prooter tivtion is not neessry, we de onstrut (pci-rep/cp) tht expressed rep78 diretly fro CMV prooter insted of the p5 prooter. As shown in Figure 3, the pci-rep/cp plsid ontins the AAV Rep nd Cp sequenes without the ITRs long with CMV prooter regulting rep78, the AAV p19 prooter regulting rep52, nd the AAV p0 prooter regulting the p gene. The two plsids pci-rep/cp nd psaav-cb-egfp were trnsfeted into Hel ells nd susequently infeted with denovirus,, or ok infeted with edi only. Hirt DNA ws extrted fro the eh ulture ondition nd genoe replition ws oserved y Southern lot. Figure 3 shows nds oserved orresponding to 1k DNA ldder. In the presene of denovirus, vetor DNA n e oserved in ultiple replitive fors. With the ddition of, replited vetor genoe ws oserved s onoer nd dier. This dt indites tht in the presene of, ws ple of repliting the vetor sequene. We lso nlyzed the Rep nd Cp proteins expression profile using western lot nlysis. As expeted, Rep protein (Figure 3) ws oserved in ll sples ut Cp protein ws only detetle in sples infeted with denovirus. This result lso indites tht lone is not suffiient for genoe replition sine when this protein ws present in ok-infeted sples, genoe replition ws not oserved. long with or denovirus is essentil for vetor genoe replition. -Rep/Cp n replite the vetor sequene nd produe raav Although vini ws le to replite the vetor genoe in the experient with CMV driven expression of P5 rep protein, raav prodution ws not deteted (dt not shown). We suspeted this ws due to the lk of psid proteins, whih were still under the ontrol of the endogenous AAV P0 prooter. We hypothesized tht we would e le to produe AAV vetors when prooter ws used to drive the expression of ll AAV proteins. As shown in Figure, AAV rep nd p (-W5 nd -W8) re under ontrol of the vini p7.5 prooter. 12 The plsid psaav-cb-egfp ws trnsfeted into HeL ells nd susequently infeted with the two es ontining AAV Rep nd Cp (-W5 nd -W8). Serving s ontrols, ultures were either infeted with denovirus or trnsfeted with the ini denovirus plsid pfdelt6. In the ells tht were oinfeted with denovirus nd -W5 nd -W8, we oserved siilr level of AAV prodution s the ells otrnsfeted with the ini denovirus plsid pfdelt6 nd -W5+-W8 infetion, t 1 10 AAV prtiles/ell nd AAV prtiles/ell respetively. In ontrst, raav vetors re still produed in the presene of -W5+-W8 t AAV prtiles/ell, leit less thn tht of denovirus infetion

3 Helper funtion of vini virus in the AAV life yle pgl3-p5-lui p5 Luiferse 3 pgl3-p19-lui p19 Luiferse pgl3-p0-lui p0 Luiferse Fold hnge enovirus Vini virus Fold hnge 0 P5 P19 P0 P5 P19 P enovirus Vini virus Figure 2 Vini virus nnot tivte deno-ssoited virus (AAV) prooters. () Hel ells were trnsfeted with firefly luiferse reporter plsids pgl3-p5-lui, pgl3-p19-lui, nd pgl3-p0-lui, whih re under the ontrol of the AAV P5, P19, or P0 prooters respetively. These ells were then infeted with denovirus or vini virus or ok infetion. Cells were hrvested t 8 hours nd then ssyed for firefly luiferse tivity. Prooter tivtion of the P5, P19, nd P0 prooters ws deteted y luinesene output nd plotted s fold hnge opred to uninfeted ontrol in hel () nd CV1 () ells. pcl-rep/cp CMV 8 k k Rep P0 Cp VP3 nd Cp proteins expression profile using western lot nlysis s previously desried. As expeted, Rep nd Cp protein (Figure d) were oserved in ll sples. This dt indites tht is le to filitte replition of the vetor genoe s well s iding in virion pkging. Siilr results were otined when single strnded vetor ws generted insted of the self opleentry vetor (dt not shown). Previous experients using n AAV8 psid yielded siilr results. 12 Experients using different ITR nd Rep serotypes were not perfored s ost reoinnt AAV vetors re AAV2 with pseudotyped psid. 1 k Rep68 Rep0 Figure 3 Vini virus n replite the genoe in the presene of. () HeL ells were trnsfeted with pci-rep/cp, pitured, nd psaav-cb-egfp. Upon trnsfetion, ells were infeted with denovirus () or vini virus () or ok infetion (ok) nd were hrvested t 8 hours post-infetion. () Southern lotting ws perfored ording to stndrd proedures for DNA trnsfer nd hyridized with 32P-leled GFP proe. The nuers on the left indite the rker positions for 1,, nd 8 k nd the () indites the position of the onoer. () Cell lyste ws prepred fro olleted ells nd protein ws extrted ording to stndrd proedure nd proed for Rep nd Cp protein. The Rep nd Cp expression profile ws oserved. (Figure ). Therefore, y ypssing the need for AAV prooter tivtion, delivering the neessry rep nd p proteins long with the ws suffiient for vetor prodution. In order to evlute the ility of -Rep/Cp to replite vetor DNA, we perfored Southern lot nlysis of the ove sples. Figure shows tht AAV genoe replition is oserved in ll sples tht were infeted with -Rep/Cp. This result is onsistent with previous oservtions s well. We lso nlyzed the Rep Vini replition ids in raav prodution These results indited tht lthough is not typil helper, it is still suffiient in order to replite nd pkge raav. In order to further evlute these helper funtions, we eployed utnt, Modified Vini Ankr (MVA). MVA ws derived y seril pssges of CVA derovini in hiken eryo firolsts nd is not perissive for replition in ost ell lines. During this proess, MVA lost pproxitely 15% of its genoe nd nuer of virulent genes resulting in hnge in plque orphology. 13 In nonperissive ell lines, MVA only produes iture virl prtiles. 1,15 HeL ells were infeted with r-aav-egfp to deliver the AAV vetor sequene s previously shown. 12 These ells were susequently infeted with -W5 nd -W8, eh t ultipliity of infetion (MOI) = 1, or MVA-W5 nd MVA-W8, eh t MOI = 20. Previous unpulished experients deterined tht lower MOI of MVA did not result in infetive AAV vetors. Figure 5 shows the raav otined fro infetion with vini (-W5 nd -W8) or MVA (MVA-W5 nd MVA- W8). Cells infeted with MVA produed signifintly fewer vetors s opred to those infeted with with AAV prtiles/ell nd AAV prtiles/ell respetively. In ontrst to the vetor yield, there ws suffiient Rep protein produed (Figure 5) nd AAV genoe replition (Figure 5) s opred to ells infeted with. This dt indites tht MVA y e suffiient for AAV protein expression nd replition, ut dditionl vini ftors issing in MVA y e essentil for raav prodution. Moleulr Therpy Methods & Clinil Developent (2015) 150

4 Helper funtion of vini virus in the AAV life yle -W5 + only pf 6 + -W8 + -W5 + -W8 -W5 + -W8 pfd6 + -W5 + -W8 8 k k d + only pf only pf k VP3 Figure -Rep/Cp n replite the vetor sequene nd produe reoinnt deno-ssoited virus (raav). () HeL ells were trnsfeted with psaav-cb-egfp s reporter nd the helper plsid pfdelt6 or pci s ontrol. Upon trnsfetion, ells were infeted with vini viruses ontining AAV Rep nd Cp sequenes under the ontrol of the vini p7.5 prooter (-W5 nd -W8 s pitured). Cells were dditionlly infeted with denovirus s positive ontrol. () Sples were hrvested nd sujeted to three rounds of freezing nd thwing nd het intivted efore eing used to trnsdue 16,095 ells. raav prodution ws visulized y oservtion of EGFP expression nd positive ells were ounted. Left pnel psaav-b- EGFP + pci + -W5 + -W8 + enovirus; Middle pnel psaav-b-egfp + pci + -W5 + -W8; Right pnel psaav-b-egfp + pfdelt6 + -W5 + -W8. () Southern lotting ws perfored ording to stndrd proedures for DNA trnsfer nd hyridized with 32P-leled GFP proe. The nuer on the left indites the rker positions for 1,, nd 8 k nd the () indites the position of the onoer. (d) Cell lyste ws prepred fro olleted ells nd protein ws extrted ording to stndrd proedure nd proed for Rep nd Cp protein. The Rep nd Cp expression profile ws oserved. -V5 + -W8 -V5/-W8 MVA-V5 + MVA-W8 MVA-V5/MVA-W8 Control 8 k k Control -V5/-W8 MVA-V5/MVA-W8 Control Figure 5 Vini replition ids in reoinnt deno-ssoited virus (raav) pkging. HeL ells were trnsdued with -AAV-eGFP s reporter nd either -W5 nd -W8 or MVA-W5 nd MVA-W8 or no vini virus s ok. () Sples were hrvested nd sujeted to three rounds of freezing nd thwing nd het intivted efore eing used to trnsdue ells. raav prodution ws visulized y oservtion of EGFP expression nd positive ells were ounted. () Southern lotting ws perfored ording to stndrd proedures for DNA trnsfer nd hyridized with 32P-leled GFP proe. The nuer on the left indites the rker positions for nd 8 k nd the () indites the position of the onoer. () Cell lyste ws prepred fro olleted ells nd protein ws extrted ording to stndrd proedure nd proed for Rep protein. The Rep expression profile ws oserved. Moleulr Therpy Methods & Clinil Developent (2015) 150 DISCUSSION Vini is virus tht undergoes its entire life yle in the ytopls of the host ell. A vst jority of its gene produts n only funtion in the ytopls s well. In ontrst, AAV is nuler virus where the replition nd pkging ll tke ple in the nuleus. Well-hrterized AAV helper viruses suh s denovirus or herpes virus re ll nuler viruses. Genes of helper viruses identified to support AAV replition nd pkging suh s E1, EORF6, et. eh hve their funtions in the nuleus. Nevertheless, ytoplsi hs een widely ited s helper virus for AAV Therefore, this wrrnted further lrifition of its role in AAV life yle. In this study, we lerly showed tht ould tivte none of the AAV prooters. This is understndle sine gene produts re inly loted in the ytopls. It is unlikely tht vini ould indue soe ellulr ftors to tivte AAV P5, P19, or P0 prooters. Despite its inility to tivte AAV prooters, hs soe hrteristis of helper virus. It hs the pility to replite the AAV genoe in the presene of nd properly pkge the AAV virion when P19 nd p0 produts were expressed, whih hs lso een reported in nother study. 20 One of the hllrks of helper for AAV replition is the ility to id in replition of the vetor genoe. Studies hve shown tht in ddition to Rep proteins nother ftor is needed in order to filitte replition. While reserh indites tht or Rep68 is needed for AAV replition, 21 denovirus infetion 22 or HSV infetion 23,2 re lso essentil for genoe replition. Siilrly, we found tht is ple of repliting the AAV genoe ut unlike its denovirus nd Herpes virus ounterprts, it nnot oplete ll funtions of helper king it suhelper for the AAV life-yle. The differene etween wild-type vini nd MVA in AAV pkging further onfirs this onlusion.

5 While it hs een previously reported tht is helper for AAV replition, 9 the SV0-trnsfored NB-E ells were used to oe to this onlusion. SV0 hs een found to provide wek helper funtion to AAV in the sene of denovirus. 25,26 SV0 T ntigen hs lso een found to intert with AAV. This intertion y e t lest prtilly responsile for SV0-edited expression of AAV2 Rep proteins. 1 Overll, with soe inil ssistne n t s wek helper for AAV replition. MATERIALS AND METHODS Cell lines HeL S3 ells were ultured in Duleo s odified Egle s ediu suppleented with 10% fetl ovine seru, 100 μg of peniillin/l, nd 100 U of streptoyin/l (Invitrogen, Crlsd, CA). CV-1 ells were ultured in iniu essentil edi with 10% fetl ovine seru, 100 μg of peniillin/ l, nd 100 U of streptoyin/l. 16,095 ells ( hun firolst ell line fro the Coriell Institute) were ultured in iniu essentil edi with 10% fetl ovine seru, 100 μg of peniillin/l, nd 100 U of streptoyin/ l. Chiken eryo firolst ells otined fro Aerin Type Culture Colletion (CRL-12203) were ultured in Duleo s odified Egle s ediu suppleented with 10% fetl ovine seru, 100 μg of peniillin/l, nd 100 U of streptoyin/l (Invitrogen). All ells were intined in huidified 37 C inutor with 5% CO 2. Plsid onstruts The plsid pgl3-si (Proeg, Mdison, WI) ws digested with MluI nd NheI (NEB, Ipswih, MA). The digested plsid ws used s the kone to onstrut ll plsids hving the AAV prooters driving the firefly luiferse gene. AAV p5, p19, nd p0 prooters were plified y polyerse hin retion y using plsid psu201 s the teplte. The plsid pci-neo ws digested with NheI nd XI (NEB). The digested plsid ws used s the kone for the pci-rep/cp plsid. The plsid ph22 ws digested with BsI (NEB) nd used s the insert for the pci-rep/cp plsid. The MVA plsid onstruts were produed y digestion of plw with BHI (NEB). The AAV Rep nd Cp sequenes were reoved fro the prb21-w5 nd prb21-w8 plsids nd loned into the plw kone. The plsids pfdelt6, p/aav, nd psaav-egfp were previously desried. 10,11 Reoinnt AAV vetor prodution raav vetors were produed y trnsfetion nd trnsdution ethods previously desried in prtiulr vini onstruts -W5 nd -W8 (ref. 12). Vetors were produed y trnsfetion using Fugene 6 (Rohe, Indinpolis, IN) t onentrtion of 1 μg per plsid per ells. HeL ells were infeted with denovirus (MOI = 10), (MOI = 1) t 8 hours post-trnsfetion. Cells were then hrvested t 8 hours post-trnsfetion. Sples underwent three rounds of freezing nd thwing nd deris ws reoved y entrifugtion. Viruses were intivted y heting the sples t 56 C for 5 inutes nd the ell lyste ws used to trnsdue 16,095 ells. GFP ws visulized with Nikon irosope using fluoresein isothioynte filter. Luiferse ssy The p5-, p19-, nd p0-luiferse onstruts were trnsfeted into HeL ells t onentrtion of 1 μg per ells. The ells were then infeted with denovirus,, or ok infetion t 8 hours post-trnsfetion. The ells were olleted t 8 hours post-trnsfetion nd lysed y the ddition of lysis uffer (100 ol/l potssiu phosphte, ph 7.8, nd 1 ol/l dithiothreitol (DTT)) to the ell pellet nd undergoing three rounds of freezing nd thwing. Cell lyste ws diluted in ssy uffer (25 ol/l glyylglyine, ph 7.8, 15 ol/l potssiu phosphte, ph 7.8, 15 ol/l MgSO, ol/l EGTA, 2 ol/l ATP, nd 1 ol/l DTT). After the ddition luiferin solution (1 ol/l D-Luiferin (Goldio, St. Louis, MO), 25 ol/l glyylglyine, ph 7.8, nd 10 ol/l DTT), light output ws red using luinesene reder. Western lot nlysis Totl proteins were extrted with lysis uffer, whih onsisted of 50 ol/l Tris t ph 8.0, 150 ol/l NCl, 1% Triton X-100, 10 ol/l DTT, nd 1 protein inhiitor (Rohe). Cell lystes were resolved on 10% sodiu dodeyl sulfte polyrylide gel eletrophoresis (SDS-PAGE) gel nd trnsferred to Helper funtion of vini virus in the AAV life yle nitroellulose erne (Bio-Rd, Herules, CA). After loking the erne with 5% nonft dry ilk in TBST uffer, whih ontins 25 ol/l Tris-HCl t ph 8.0, 150 ol/l NCl, nd 0.1% Tween20, the erne ws inuted with the priry ntiody, nti-aav psid (B1, Aerin Reserh Produts, Belont, MA) or nti-aav Rep (303.9, Aerin Reserh Produts) t dilution of 1:500 t C overnight. The erne ws wshed nd inuted with horserdish peroxidse-onjugted sheep nti-ouse IgG ntiody (Sig, St. Louis, MO) t dilution of 1:2,000. The erne ws developed using n enhned heiluinesent sustrte (Aersh- Phri Bioteh, Pistwy, NJ). Southern lotting hyridiztion Alkline grose gel eletrophoresis ws used to nlyze DNA replition of the AAV genoe s desried previously. 27 Briefly, HIRT DNA ws extrted using stndrd ethods nd 10 μg of DNA ws resolved on 1% lkline grose gel. After gel lotting, the erne ws hyridized with 32P-leled proe tht ws produed using frgent speifi to the EGFP portion of eh vetor. The purified frgent ws leled y [α-32p] dctp using Prie-IT II Rndo Prier Leling Kit (Strtgene, L Joll, CA). The erne ws nlyzed y X-ry utordiogrphy. Reoinnt MVA prodution MVA virus nd plsid plw ws kindly provided y Bernrd Moss. To generte MVA, Chiken eryo firolst ells were seeded into six-well plte nd infeted with MVA t n MOI of The ells were susequently trnsfeted with the trnsfer plsid 1.5 hours following infetion with Fugene per nufturer instrutions. The ells were inuted overnight t 37 C in 5% CO 2 nd the edi ws repled. At ~8 hours postinfetion the ells were hrvested in 1 l of edi nd the sple underwent three rounds of freezing nd thwing. The resulting lyste ws used to reinfet Chiken eryo firolst ells nd plque purified for severl rounds s previously desried. 28 CONFLICT OF INTEREST The uthors delre no onflit of interest. ACKNOWLEDGMENT This study ws supported y Ntionl Institutes of Helth (NIH) (R01HL08381 nd R01HL11152 to W.X.); NIH under Ruth L. Kirshstein Ntionl Reserh Servie Awrd T32-HL fro the Ntionl Hert Lung nd Blood Institute (to A.R.M.). Referenes 1. Muzyzk, N (1992). Use of deno-ssoited virus s generl trnsdution vetor for lin ells. Curr Top Miroiol Iunol 158: Crter, BJ, Antoni, BA nd Klessig, DF (1992). enovirus ontining deletion of the erly region 2A gene llows growth of deno-ssoited virus with deresed effiieny. Virology 191: Nsh, K, Chen, W nd Muzyzk, N (2008). Coplete in vitro reonstitution of denossoited virus DNA replition requires the inihroosoe intenne oplex proteins. J Virol 82: Sulski, RJ nd Shenk, T (1988). enovirus E1B 55-Mr polypeptide filittes tiely ytoplsi uultion of deno-ssoited virus RNAs. J Virol 62: Xio, X, Li, J nd Sulski, RJ (1998). Prodution of high-titer reoinnt denossoited virus vetors in the sene of helper denovirus. J Virol 72: Zhou, X nd Muzyzk, N (1998). In vitro pkging of deno-ssoited virus DNA. J Virol 72: Csto, BC, Athison, RW nd Hon, WM (1967). Studies on the reltionship etween deno-ssoited virus type I (AAV-1) nd denoviruses. Virology 32: Ogston, P, Rj, K nd Berd, P (2000). 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