Development of Patient-specific AAV Vectors After Neutralizing Antibody Selection for Enhanced Muscle Gene Transfer

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1 The Americn Society of Gene & Cell Therpy originl rticle Development of Ptient-specific AAV Vectors After Neutrlizing Antibody Selection for Enhnced Muscle Gene Trnsfer Chengwen Li,2, Shuqing Wu,3, Blke Albright, Mtthew Hirsch, Wuping Li 4, Yu-Shn Tseng 5, Mvis Agbndje-McKenn 5, Scott McPhee 6, Arvind Asokn,7 nd R Jude Smulski,8 Gene Therpy Center, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA; 2 Deprtment of Peditrics, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA; 3 Chin Ntionl Acdemy of Nnotechnology & Engineering, Tinjin, Chin; 4 Institute of Pthogen Biology, Chinese Acdemy of Medicl Sciences, Beijing, Chin; 5 Deprtment of Biochemistry nd Moleculr Biology, Center for Structurl Biology, McKnight Brin Institute, University of Florid, Ginesville, Florid, USA; 6 Asklepios BioPhrmceuticl Inc., Chpel Hill, North Crolin, USA; 7 Deprtment of Genetics, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA; 8 Deprtment of Phrmcology, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA A mjor hindrnce in gene therpy trils with denossocited virus (AAV) vectors is the presence of neutrlizing ntibodies (NAbs) tht inhibit AAV trnsduction. In this study, we used directed evolution techniques in vitro nd in mouse muscle to select novel NAb escpe AAV chimeric cpsid mutnts in the presence of individul ptient serum. AAV mutnts isolted in vitro escped brod ptient-specific NAb ctivity but hd poor trnsduction bility in vivo. AAV mutnts isolted in vivo hd enhnced NAb evsion from cognte serum nd hd high muscle trnsduction bility. More importntly, structurl modeling identified 00 mino cid motif from in vrible region (VR) III tht confers this enhnced muscle tropism. In ddition, predominntly cpsid bet brrel templte with specific preference for AAV/ in VR VII locted t threefold symmetry xis fcilittes NAb escpe. Our dt strongly support tht chimeric AAV cpsids composed of modulr nd nonoverlpping domins from vrious serotypes re cpble of evding ptient-specific NAbs nd hve enhnced muscle trnsduction. Received 2 Februry 205; ccepted 8 July 205; dvnce online publiction 25 August 205. doi:0.038/mt INTRODUCTION Adeno-ssocited virus (AAV) vectors hve been explored extensively in numerous preclinicl studies nd number of these studies re currently trnslting into encourging phse, 2, nd 3 clinicl trils. 5 While AAV gene therpy continues to yield clinicl results supportive of the hope for eventul tretment of mny diseses, the presence of ptient neutrlizing ntibodies (NAbs) remins chllenge. NAb-medited elimintion of AAV vectors hs become rte-limiting step in dvncing the field nd determinnt for repet dministrtion of AAV gene trnsfer.,2,6 The fct tht more thn 90% of the popultion hs been exposed to nturl infection, nd hlf of those infected crry NAbs in their blood, 7 7 highlights the significnce of this problem. Although vectors re used in the mjority of clinicl trils, 2,8,9 other serotypes, such s AAV, 8, or customized vectors such s.5 re being explored.,6,20 In n erly phse clinicl tril with hemophili B ptients using n vector encoding the fctor IX (F.IX) trnsgene, ~0% of norml F.IX levels were chieved in one ptient who lcked NAbs nd no detectble F.IX ws observed in second ptient with preexisting NAbs. 2 More recently, we performed phse clinicl tril in six ptients with Duchenne musculr dystrophy (DMD) using the chimeric.5 vector encoding mini-dystrophin trnsgene for muscle delivery. Before intrmusculr (i.m.) injection, no NAbs were found in three ptients, low NAb titers were detected in one ptient, nd high NAb titers were detected in the remining two ptients. After injection, vector genomes were only detected in muscle biopsies from ptients with no or low titers of NAbs. 6 This observtion suggests tht NAbs inhibit AAV trnsduction following direct i.m. injection. Severl pproches to overcome AAV NAbs include: plsmpheresis, 2 reduction of NAb production with B-cell depletion, 22 use of empty AAV cpsid decoys, 23 lterntive AAV serotypes tht re nturlly resistnt to NAbs, 9,24 27 selection of NAb escpe mutnts from n error-prone PCR librry, site-directed modifiction of AAV cpsid proteins, 3 nd AAV cpsid-ssocited polymers tht prevent NAb recognition Bsed on kinetic nlysis of the NAbs in our phse clinicl tril with hemophili ptients, NAb cross-rectivity ws suggested s primry mechnism of neutrliztion of unrelted AAV serotypes. 7 This observtion ws confirmed in results from our DMD phse clinicl tril. After i.m. injection of chimeric.5, ll three ptients initilly lcking NAbs hd NAb titers t :00 (ref. 6). They lso displyed differentil inhibition to vrious AAV serotypes. Regrding the AAV cpsid modifiction strtegies to develop AAV mutnts for NAb evsion, Mheshri et l. 30 used n librry derived Correspondence: Chengwen Li, Gene Therpy Center, 79 Thurston-Bowles, CB 7352, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin 27599, USA. E-mil: chengwen@med.unc.edu nd R Jude Smulski, Gene Therpy Center, 79 Thurston-Bowles, CB 7352, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin 27599, USA. E-mil: rjs@med.unc.edu Moleculr Therpy vol. 24 no., jn

2 The Americn Society of Gene & Cell Therpy from error prone PCR to isolte AAV NAb escpe mutnts in the presence of rbbit nti- ser in vitro. Other studies, including ours, hve selected mutnts with high tissue tropism using n AAV librry from DNA shuffling of different serotypes; however, NAb evsion bility ws only tested postselection in cell lines or in the presence of humn intrvenous immunglobin In the current study, we isolted AAV NAb escpe mutnts in the presence of individul ptient serum from our DMD clinicl tril using AAV directed evolution in vitro. Most importntly, we lso performed the selection of AAV mutnts with NAb evsion in vivo to determine whether the mutnts isolted from one ptient s serum re ble to escpe NAb ctivity from other ser nd retin high tissue trnsduction. In HEK293 cells, AAV mutnts selected from individul ptient serum escped NAb ctivity in ser from ll subjects; however, ll in vitro selected mutnts were compromised in trnsducing trget tissue in vivo. Importntly, we lso selected AAV mutnts with NAb evsion in vivo in BALB/c mouse muscle to determine whether the mutnts isolted from one ptient s serum escped NAb ctivity from other ptient ser. The in vivo selection resulted in severl cpsid mutnts recovered from skeletl muscle tht not only escped NAbs but lso displyed high muscle trnsduction efficiency compred to most nturlly occurring AAV serotypes. To explore the cpsid protein mino cid () sequence required for NAb evsion nd enhnced tropism, we conducted sequence lignment nd structurl nlysis studies. Phylogenetic nlysis nd structurl modeling of severl of the selected mutnts helped to identify AAV cpsid motifs tht cn be further engineered to modulte NAb evsion while retining muscle tropism. This pproch of generting personlized, ptient-specific NAb AAV escpe mutnts represents prdigm shift in steps required when exploiting AAV directed evolution to overcome immune limittions currently impcting gene trnsfer studies. RESULTS Chrcteriztion of AAV NAb escping mutnts isolted from HEK293 cells In our phse DMD clinicl tril, six ptients were treted vi i.m. injection with chimeric.5 encoding the mini-dystrophin trnsgene. 6 Prior to vector dministrtion, three ptients hd no NAbs; however, ser collected 00 dys posttretment showed tht NAb titers incresed to :00 6. To select AAV NAb escpe mutnts, undiluted ser from these ptients were combined with n AAV cpsid shuffled librry 35,38 nd pplied to HEK293 cells in the presence of denovirus (Ad). Forty-eight hours posttretment, cell lystes contining the NAb escpe mutnts were gin used to infect HEK293 cells in the presence of Ad for second cycle of AAV mplifiction. After four rounds of mplifiction, NAb AAV mutnts were isolted nd sequenced. Nine, 8, nd 2 mutnts were isolted from ptient ser, 2, nd 3, respectively (Supplementry Figures S). Most mutnts from ser nd 2 contined chimeric VP3 of nd (Tble nd Supplementry Figure SA,B). Eleven mutnts isolted from serum 3 contined the VP3 C-terminl from (Tble nd Supplementry Figure SC), nd nine of them hd the VP3-N-terminl of AAV. In ddition to cpsid composition from different serotype of AAV, ll mutnts contined t lest one point muttion. Most mutnts produced comprble virus yields to, but lower titers were produced by mutnts AAV00-2 nd 5, AAV002-9, nd AAV003-9 (Tble ). The AAV00-2 nd 5 mutnts hve proline residues, L636P nd N737P, respectively, close to VP interfces, nd the G640E muttion in AAV002-9 is in buried position inside the cpsid likely to cuse steric hindrnce tht my prevent virl cpsid ssembly. For AAV003-9 the T233Q residue is locted in β-strnd inside the cpsid. Thus, while the mechnism(s) behind reduced or lck of virus production is uncler, it is likely tht the point muttions in these constructs chnged the VP structure, lthough individul AAV VP subunit cn be formed, it is possible tht the muttions result in insufficient AAV virion ssembly from 60 VP subunits. After identifiction nd virus production in HEK293 cells, recombinnt mutnts pckging firefly luciferse trnsgene (luc) were tested for their bility to trnsduce HEK293 cells. The mutnts possessed different trnsduction efficiency s compred with (Tble ). Lower trnsduction ws demonstrted for the mutnts with the exception of mutnt AAV00-4, which hd similr trnsduction bility to. NAb escping bility of the mutnts isolted in HEK293 cells To investigte the cpcity of mutnts recovered from HEK293 cells to escpe NAbs from individul ptient serum, we incubted mutnts with undiluted serum t 4 C for 2 hours. The unbound virus trnsduction ws determined by dding the AAV mutnt/ serum mixture to HEK293 cells. For escpe mutnts from serum, ll tested, with the exception of AAV00-7, exhibited trnsduction >50% of control without serum, especilly mutnts AAV00-, 4, nd 5 which showed >80% trnsduction (Tble nd Supplementry Figure S2). This my be becuse the N-terminus of AAV00-7 VP3 ws derived from AAV, nd AAV trnsduction ws inhibited by serum. In contrst, the other eight mutnts VP3 ws derived from nd 9, nd nd 9 trnsduction ws not inhibited to the sme extent s ws AAV. Among the escpe mutnts from serum 2, diverse cpsid composition ws observed. Bsed on NAb escpe nlysis, mutnts AAV002-9, 4, nd 5 were resistnt to serum 2, wheres the other five mutnts were strongly inhibited (Tble nd Supplementry Figure S2). Among the inhibited mutnts, AAV002-6 ws derived entirely from the cpsid but contined two muttions, S292L nd K556E. It is interesting to note tht serum 2 lso inhibited trnsduction from mutnts AAV002- nd 3, composed of n AAV VP N-terminus (VPu) nd nd 9 VP3, while wild-type (wt) nd 9 escpe the NAb ctivity of serum 2. This suggests the involvement of the VPu in ntibody recognition. Also, lthough serum 2 completely suppressed trnsduction nd prtilly inhibited AAV trnsduction, mutnt AAV002-4, chimer of AAV nd 2, trnsduced HEK293 cells efficiently in the presence of serum 2. This is likely due to the presence of sequence vrition t or close to ntigenic regions of AAV nd. 3,39 In the presence of serum 3, trnsgene expression ws decresed for AAV, 2, 4, 8, nd 9 s well s chimeric.5. Among the 2 mutnts, AAV003-7 nd 9 were ble to escpe the NAb ctivity to induce trnsgene expression >50% of control (Tble nd Supplementry Figure S2). The other isolted mutnts could not escpe the NAb ctivity of serum vol. 24 no. jn. 206

3 The Americn Society of Gene & Cell Therpy Tble Chrcteriztion of AAV mutnts isolted from HEK293 cells Composition of HEK293 Virus titer b cpsid c VP3 composition Serum 2 Nb ctivity Serum 3 Nb ctivity Serum AAV AAV AAV AAV AAV AAV00-2 NC AAV AAV00-5 NC AAV AAV AAV AAV AAV002-9 NC AAV AAV AAV AAV AAV AAV AAV AAV AAV AAV AAV AAV AAV AAV AAV AAV003-9 NC AAV with 5 muttions 2 with 5 muttions AAV AAV, deno-ssocited virus; NC, not comprble due to lower virus titer; PBS, phosphte-buffered sline. Fold chnge when compred to trnsduction, ( ) indictes fold decrese. b Virus titer yield (0 7 prticles/µl) in ml of superntnt from one 0 cm plte from one trnsfection. c The composition of cpsid derived from wild-type AAV in the sequentil order. d Serum neutrlizing ctivity ginst AAV trnsduction when undiluted ser were incubted with AAV vectors. ( ) indictes AAV trnsduction is >50% when compred to AAV incubtion with PBS; (+) AAV trnsduction <50% of control group. Cross-rectivity of ser from ptients ginst AAV mutnts isolted in HEK293 cells A number of mutnts isolted from ptient serum hd the bility to escpe the NAb ctivity of their cognte serum s described bove. Next, we nlyzed the cross-rectivity of ptient NAbs with mutnts isolted ginst other ptient ser. Some mutnts isolted from one serum could lso escpe the NAb ctivity of the other two ser, such s AAV00-,, 2, 4, 7, AAV002-9, 4, nd AAV003-9 (Tble nd Supplementry Figure S2). These results indicte tht it is possible to isolte AAV mutnts tht possess generic bility to evde NAbs from multiple subjects. Other mutnts hd n bility to escpe the NAbs from one but not nother lterntive ptient serum such s AAV00-4, 5, 5, AAV002-5, nd AAV Interestingly, most mutnts isolted from serum 3 could only evde NAbs of Moleculr Therpy vol. 24 no. jn

4 The Americn Society of Gene & Cell Therpy Mus-3 V25L AAV 2 N34S 348 E49D 447 E532K R726H 737 AAV N34S/E362Q/D385N E533K 728 R727H 738 Mus-4 Mus-9 AAV3 83 G2D 233 A274T E533K/G72S/K727H/N737P 738 AAV Mus- Mus-2 Mus T425Q P20S 37 E532K/R726H/N736P 737 A39T 40 R69K/L89I N384T E532K/R726H/N736P 737 AAV AAV Mus G720S/R726H/N736P D78G D348E E53K/R725H/N735P 736 Mus-24 AAV AAV AAV AAV b AAV c 0,000,000 mus9 mus mus2 mus9 Luc expression (photon/seconds),000,000 00,000 0,000,000 AAV mus9 mus mus2 mus9 Figure Trnsduction efficiency of AAV mutnts recovered from mouse skeletl muscle. 0 0 prticles of the AAV shuffled librry virus incubted with humn serum 3 nd dministered vi i.m. injection to 6-week-old femle BALB/c mice. Three dys postinjection, Ad dl309 ws injected into the sme muscle. () After 2 dys, AAV mutnt cpsids were recovered from the injected muscle nd sequenced. These mutnt cpsids were used to pckge the luc trnsgene. 0 9 prticles of AAV/luc were dministered vi i.m. injection in both legs of 6-week-old femle BALB/c mice (n = 2 mice). One week post AAV ppliction, (b) imging ws crried out nd (c) the photon signl ws mesured nd the verge signl from ll four legs ws clculted. The exposure time for imging ws 0 seconds for, Mus3, nd 2, nd 5 minutes for other mutnts nd AAV serotypes. AAV, deno-ssocited virus; i.m., intrmusculr. serum 3 but could not evde NAbs from serum or 2 (Tble nd Supplementry Figure S2). These dt suggest tht AAV cpsid NAb escpe mutnts isolted from individul serum my be different from those from trditionl pooled ser pproch. In vivo trnsduction profile of AAV mutnts isolted in HEK293 cells To determine the tissue tropism of AAV mutnts isolted in HEK293 in the presence of ptient serum, 0 prticles of selected AAV vectors encoding the luc trnsgene were retroorbitlly injected into 6-week-old femle BALB/c mice. After week, in vivo imges were cptured. Consistent with the literture, nd 2 minly trnsduced the liver, AAV4 trnsduced the hert, AAV trnsduced both liver nd hert, nd trnsduced lmost every tissue (Supplementry Figure S3). Compred to prentl AAV serotypes, ll mutnts except for AAV002-4 trnsduced the liver, but to lesser extent thn. Two weeks postinjection, tissue ws hrvested nd luciferse ctivity ws nlyzed. AAV003-7, chimeric of AAV nd 2, hd similr muscle trnsduction to serotypes AAV4 nd 9, higher thn prentl serotypes (Supplementry Figure S3). Overll lower trnsduction ws observed cross different tissues using chimeric mutnts compred to prentl serotypes. These results suggest tht mutnts with the bility to escpe NAb 56 vol. 24 no. jn. 206

5 The Americn Society of Gene & Cell Therpy AAV.5 mus mus2 mus9 PBS Serum b 0,000,000 PBS Serum c Luc expression (photon/seconds),000,000 00,000 0,000 Inhibition efficiency (%) ,000 0 AAV.5 mus mus2 mus9 AAV.5 mus mus2 mus9 Figure 2 NAb escpe potentil for AAV mutnts derived from muscle. 0 9 prticles of AAV/luc from mutnts isolted from mouse muscle nd wild serotypes were incubted with n equl volume of fivefold diluted ptient serum 3 or PBS for 2 hours t 4 C, then dministered vi i.m. injection to muscles of both legs in 6-week-old femle BALB/c mice (n = 2). Three weeks post AAV ppliction, mice were imged for () luc expression. The photon signl ws mesured, nd (b) the verge signl for mice treted with humn serum 3 or PBS ws clculted for 4 legs. (c) The inhibition of humn serum 3 on AAV trnsduction ws nlyzed by comprison of trnsgene expression in serum-treted mice over tht of PBS-treted mice. The exposure time for imging ws 0 seconds for, Mus3, nd 2, nd 5 minutes for other mutnts s well s nd 9. AAV, deno-ssocited virus; i.m., intrmusculr; NAb, neutrlizing ntibody; PBS, phosphte-buffered sline. should be further nlyzed in vivo for mintennce of tissuespecific trnsduction. Isoltion of AAV mutnts from mouse skeletl muscle in the presence of humn serum As proof of principle, we successfully obtined chimeric cpsid AAV mutnts tht evde NAb ctivity of humn ser in HEK293 cells. Although most of these mutnts trnsduced cells efficiently in vitro, this my not trnslte into successful trnsduction in vivo. To develop tissue-tropic AAV mutnts with NAb evsion bility, we directly injected mixture of the AAV shuffling librry nd DMD ptient 3 serum into the muscle of 6-week-old femle BALB/c mice. Three dys postinjection, Ad ws injected into the sme muscle to mplify the AAV genomes. Two dys post Ad injection, muscle ws collected for totl DNA extrction. PCR ws used to mplify mutnt cpsid sequences, nd PCR products were subsequently cloned into the pxr2 bckbone. Eight colonies were obtined nd sequenced; every clone contined the C-terminus of the VP3 cpsid except for Mus4 for which the C-terminus contined 0 residues from (Figure ). Trnsduction efficiencies of AAV mutnts recovered from mouse skeletl muscle To determine the reltive muscle tropism of chimeric cpsid AAV mutnts isolted from mouse muscle, 0 9 prticles of AAV mutnts s well s prentl AAV serotypes encoding the luc trnsgene were delivered vi i.m. injection of 6-weekold femle BALB/c mice. One week postinjection, imging ws performed. As shown in Figure, remined superior for muscle trnsduction, with trnsduction efficiency order: >Mus2>Mus3>Mus>=Mus4=Mus9=Mus24= AAV>>>Mus4>Mus9. To exmine muscle trnsduction fter systemic dministrtion, 0 prticles of AAV mutnts s well s prentl serotypes were injected vi the retro-orbitl vein into 6-weekold femle BALB/c mice. One week postinjection, imging ws performed. At 2 weeks postinjection, vrious tissues were collected for in vitro luc ctivity nlysis. Consistent with prior reports, ws superior in trnsducing mouse tissue (Supplementry Figure S4). Among six mutnts, Mus9 nd 24 were ble to trnsduce the liver more efficiently thn other mutnts, with similr performnce compred to prentl, nd lower efficiency thn or 9. Regrding muscle tropism, Mus ws superior in trnsducing both hert nd skeletl muscles compred to other mutnts. However, hert trnsduction of Mus ws lower thn tht of, 8, nd 9. Skeletl muscle trnsduction by Mus ws higher thn tht of but lower thn tht of or 9. Lstly, Mus9 induced better skeletl muscle trnsduction thn. These dt indicte tht high muscle trnsduction of mutnts delivered vi i.m. injection does not trnslte into high muscle tropism by systemic dministrtion. These results re similr to the observtion tht, the Moleculr Therpy vol. 24 no. jn

6 The Americn Society of Gene & Cell Therpy mus mus2 mus9 PBS Serum Serum 2 b Luc expression (photo/seconds) 0,000,000,000,000 00,000 0,000,000 mus mus2 mus9 PBS Serum c Inhibition efficiency (%) mus mus2 mus9 d Luc expression (photon/seconds) 0,000,000,000,000 00,000 0,000,000 mus mus2 mus9 PBS Serum e Inhibition efficiency (%) mus mus2 mus9 Figure 3 Cross-rectivity of NAbs on AAV mutnts isolted from muscle. 0 9 prticles of AAV/luc mutnts isolted from mouse muscle nd wild serotypes were incubted with n equl volume of fivefold diluted ptient serum, 2, or PBS for 2 hours t 4 C, then the mixture of AAV/luc nd serum ws dministered vi i.m. injection into both legs of 6-week-old BALB/c mice (n = 2). () At week post AAV dministrtion, imging ws tken for luc expression. The verge signl for legs in mice treted with (b) ptient serum or (d) 2 or PBS ws clculted (b nd d for serum nd 2, respectively). The inhibition of humn (c) serum or (e) 2 on AAV trnsduction ws nlyzed by comprison of trnsgene expression in serum treted mice over tht in PBS mice. The exposure time for imging ws 0 seconds for, Mus3, nd 2, nd 5 minutes for other mutnts nd nd 9. AAV, deno-ssocited virus; i.m., intrmusculr; NAb, neutrlizing ntibody; PBS, phosphte-buffered sline. serotype superior for muscle trnsduction following i.m. injection, performs worse thn or 9 in trnsducing muscle fter systemic ppliction. NAb escping bility of muscle-derived AAV mutnts in vivo In vitro dt showed tht undiluted ptient serum 3 inhibited trnsduction by prentl AAV serotypes, 2, 4, 8, 9, nd mutnt.5 (Tble nd Supplementry Figure S2). To determine whether the chimeric cpsid AAV mutnts isolted from muscle tissue could evde NAb ctivity from serum nd successfully trnsduce mouse muscle, we delivered 0 9 prticles of AAV mutnts or prentl serotypes encoding the luc trnsgene mixed with fivefold diluted serum 3 or phosphte-buffered sline (PBS) vi i.m. injection to 6-week-old femle BALB/c mice. One week postinjection, imging ws performed. Serum 3 inhibited muscle trnsduction >50% for ll prentl serotypes nd mutnt.5 except for, nd lso inhibited Mus2. However, no suppression of luc expression ws observed in muscle injected with Mus3, 4,, 9, or 24 (Figure 2). Cross-rective NAb escping bility of muscle-derived AAV mutnts In vitro experiments demonstrted tht the mutnts isolted from serum 3 hd higher cpcity to escpe NAb ctivity from serum 3 thn from ser nd 2. To determine whether muscleisolted mutnts tht escped serum 3 NAbs could lso evde NAbs from ser nd 2, we delivered 0 9 prticles of AAV mutnts or prentl serotypes encoding the luc trnsgene mixed with fivefold diluted serum or 2 or PBS vi i.m. injection to 6-week-old femle BALB/c mice. As shown in Figure 3, strong inhibition of trnsduction in the presence of ser or vol. 24 no. jn. 206

7 The Americn Society of Gene & Cell Therpy /8 /6/2 / b /6/2 b Luc expression efficiency (%) /8 / ,024 Serum dilution Figure 4 The C-terminus of VP3 is responsible for NAb binding. () A digrm of AAV mutnts with swpped domins between the nd cpsids. (b) NAb titer nlysis of AAV mutnts with domin swpping. Ptient 3 serum with twofold seril dilutions were incubted with AAV mutnts nd or t equl volumes of 0 8 prticles for 2 hours t 4 C, then dded to RC32 cells in the presence of Ad dl309 (MOI of 5). One dy lter, luc ctivity ws mesured, nd trnsgene expression efficiency ws clculted ginst trnsduction of AAV mixed with PBS only (control). AAV, deno-ssocited virus; MOI, multiplicity of infection; NAb, neutrlizing ntibody; PBS, phosphtebuffered sline. ws seen week postinjection. Unlike serum 3, both ser nd 2 suppressed muscle trnsduction >50%; muscle trnsduction ws not inhibited (>50% of PBS group). Wheres the Mus2 mutnt ws inhibited by serum 3, ser or 2 did not inhibit muscle trnsduction. Although serum ws ble to inhibit muscle trnsduction from the other 5 Mus mutnts >50%, serum 2 only inhibited muscle trnsduction from Mus3 nd 24, but not from Mus4,, nd 9. These results indicte tht some mutnts isolted from serum 3 lso possess NAb escping bility towrd other ser but tht the efficiency of NAb escpe vried cross ser. Elucidtion of the cpsid regions for mutnts with NAb escpe bility nd muscle tropism We successfully recovered eight mutnts from mouse muscles in the presence of humn serum, nd the cpsid sequence dt showed n exclusivity of cpsid sequence in the C-terminus from residues 446 to the end. We wondered whether this VP region might ply role in NAb escpe bility for the mutnts. We therefore swpped the corresponding domins of nd, nd subsequently crried out n in vitro NAb titer ssy. c Luc expression(photon/seconds) 00,000,000 0,000,000,000,000 00,000 0,000 /6/2 Figure 5 A cpsid domin of contributing to robust muscle tropism. 0 9 prticles of AAV/luc from mutnt 62 nd wt or 6 were dministered vi i.m. injection to muscles of both legs of 6-week-old femle BALB/c mice. One week postinjection, imging ws tken nd clculted quntittively. () Digrm of 62 mutnt. (b) Imging week post AAV/luc vector i.m. dministrtion. The exposure time ws minute for nd 62, nd 0 seconds for. (c) Quntittion of luc imging. (d) Fold increse of luc expression from 62 nd trnsduction in muscles over tht of the vector. AAV, deno-ssocited virus; i.m., intrmusculr; PBS, phosphte-buffered sline. It ws demonstrted tht the sme dilution of serum 3 inhibited nd the /2 chimeric vector trnsduction by 50% t 28-fold dilution but inhibited nd /8 t more concentrted 4-fold dilution (Figure 4). These results suggest tht it is possible to identify VP regions responsible for NAb binding vi selection of AAV mutnts in the presence of NAbs. As described bove, six out of eight Mus mutnts recovered from muscle in the presence of humn serum showed higher muscle tropism thn prentl, nd four mutnts (Mus3,, 9, nd 24) contined motif from the cpsid round residues 347 to 446 in VP. To determine whether the motif from cpsid plys role in muscle tropism, we mde n mutnt virus (62) by swpping cpsid frgment of t residues 347 to 446 into the cpsid, nd delivered 0 9 prticles vi i.m. injection to 6-week-old femle BALB/c mice. Compred to muscle trnsduction, 3- to 0-fold higher trnsgene expression ws chieved using the 62 vector. However, 62 muscle trnsduction remined sevenfold less efficient thn (Figure 5). This result suggests tht residues 347 to 446 from VP contribute to muscle tropism in recovered mutnts. Thus, in this study, we re not only ble to identify puttive cpsid motifs responsible for NAb binding, but lso determine motifs importnt for tropism nd enhnced muscle trnsduction by studying in vivo escpe mutnts. d Fold increse to /6/2 Moleculr Therpy vol. 24 no. jn

8 The Americn Society of Gene & Cell Therpy b AAV003-5 (i) AAV003-9 AAV003-3 AAV00-7 AAV003-8 AAV002-6 AAV AAV003-6 (ii) AAV003-8 AAV003- AAV003- AAV003-0 Muscle-AAV-NAB- Muscle-AAV-NAB-24 Muscle-AAV-NAB-3 Muscle-AAV-NAB-4 (iii) Muscle-AAV-NAB-9 Muscle-AAV-NAB-4 Muscle-AAV-NAB-9 AAV003-7 AAV00-4 AAV002-3 (iv) AAV002- AAV002-9 Muscle-AAV-NAB-2 AAV00- AAV00-2 AAV00-5 AAV00-7 (v) AAV00-4 AAV00-5 AAV00- AAV002-6 AAV003-9 AAV002-5 AAV AAV002-4 AAV Figure 6 Phylogeny nd structurl models of AAV mutnts. () Neighbor-joining phylogeny of the VP cpsid sequence of NAb evding AAV mutnts. Cpsid sequences of AAVs were ligned using ClustlW, nd the phylogeny ws generted using neighbor-joining lgorithm nd Poisson correction to clculte distnces. HEK293 isoltes AAV00-, AAV002-4, AAV003-9, nd the skeletl muscle isolte Mus2 were selected for further structurl modeling bsed on their distinct cldes on the phylogenetic tree nd functionl bility to brodly escpe NAbs. (b) Threedimensionl structurl models of select AAV NAb escpe mutnt tht brodly escpe NAbs. Three-dimensionl surfce models of cpsid subunit trimer/threefold nd the pentmer/fivefold symmetry xis regions of AAV NAb escpe mutnts isolted from 293 cells in vitro or recovered from mouse skeletl muscle in vivo. Amino cid residues derived from different AAV serotypes re highlighted s follows: purple (AAV), blue (), hot pink (), green (), nd brown (). Point muttions re highlighted in ornge. Specific residues nd point muttions re listed in Supplementry Figure S. The surfce model of the full cpsid (60-mer) of the muscle isolte Mus2 is lso shown with residues derived from highlighted in green nd residues derived from colored brown. All structurl models were visulized using PyMOL ( org/, The PyMOL Moleculr Grphics System, Version.5.0.4; Schrödinger). AAV, deno-ssocited virus; NAb, neutrlizing ntibody vol. 24 no. jn. 206

9 The Americn Society of Gene & Cell Therpy DISCUSSION The objective of this proof-of-principle study ws to explore the possibility of isolting AAV mutnts tht evde NAbs nd retin efficient tissue trnsduction to fcilitte repet dministrtion in ptients who were previously treted with AAV. We first isolted AAV mutnts in the presence of ser from different humn subjects in vitro nd demonstrted tht these mutnts possessed the cpcity for evding NAb ctivity from different ser. Wheres some mutnts hd the generic bility to escpe NAbs cross different serum sources, others could escpe NAbs from cognte serum fr more efficiently thn from other ser. Essentilly ll mutnt cpsids selected in vitro hd reduced trnsduction in vivo compred to stndrd serotypes. However, in selecting tissue-specific AAV mutnts with NAb evsion bility, we recovered eight mutnts tht escped humn serum to trnsduce mouse muscle. Six of the mutnts initited higher muscle trnsduction thn most serotypes. Among the six mutnts with high muscle tropism, five were better ble to evde NAbs in cognte serum thn ny prentl AAV serotype. Another hd the bility to escpe NAbs from other ser with efficiency vrying by mutnt nd serum. Finlly, bsed on our NAb escpe mutnt cpsid composition dt, we identified the domins responsible for NAb escpe ctivity nd muscle tropism. AAV gene therpy hs been successfully used for phse clinicl trils in ptients with vision nd bleeding disorders. 5 Trnsgenic cogultion F.IX hs been successfully delivered to the bloodstrem fter AAV vector delivery to the liver in ptients with hemophili B,2. However, this tretment hs only benefited smll set of ptients, s more thn 50% of people crry NAbs tht inhibit AAV trnsduction. To evde AAV NAb ctivity, severl strtegies hve been tested to either protect AAV vectors from neutrliztion or to decrese NAb titers in the ptient s blood. Polymer (e.g., polyethylene glycol) hs been used to cot the AAV surfce nd block NAb recognition This pproch is difficult to reproduce with respect to equl coting nd hs been suggested to chnge the AAV trnsduction profile. A second pproch hs used error-prone PCR or DNA shuffling to generte librry of AAV cpsid vrints to select for NAb escpe mutnts in the presence of NAbs in vitro ,37 This pproch hs yielded novel cpsids; however, it bers the potentil limittion of generting cpsids with unknown trnsduction efficiency in vivo. In the present study, we recovered escpe mutnts from HEK293 cells in the presence of humn NAb ser tht hd similrly compromised trnsduction in vivo. In niml models, severl studies hve found tht seprte serotypes of AAV show little or no NAb cross-rectivity; thus, it hs been suggested to use other serotypes to overcome NAb inhibition. 9,3,24 27 While this strtegy is logicl, dditionl studies suggest tht concern remins bout the possibility of cross-rectivity in most humns which my not be predicted in nimls. 7 A finlly ttempted pproch to resolve NAb inhibition hs been rtionl muttion of the NAb binding domin on the AAV cpsid surfce. 27 This strtegy requires informtion bout monoclonl ntibody (mab) epitopes nd the structure of the AAV virion. While successful nd informtive, this pproch is inherently limited due to polyclonl nture of humn NAbs, nd the difficulty in obtining mabs representing ll generted NAbs. In ddition to vector modifiction, severl clinicl pproches hve been employed such s plsmpheresis prior to vector delivery. 2 Due to the reltive inefficiency of ech round of plsmpheresis nd the fct tht even low titers of NAbs (<:5) cn brogte AAV trnsduction, this strtegy is generlly only suitble for ptients with lower strting titers of AAV NAbs nd requires multiple sessions. Similr to plsmpheresis, the use of blloon ctheters in combintion with sline flush hve shown efficcy for liver gene trnsduction in the presence of low to moderte NAb titers. 40 More recently, the use of n nti-cd20 ntibody (rituximb) to chieve B-cell depletion for 6 9 months hs been reported. This pproch is not directed t (ntibody-producing) plsm cells nd is effective in reducing AAV NAb in only minority of subjects who hve NAb titer less thn :,000 (ref. 22). A finl clinicl pproch pplies excessive empty AAV cpsids s decoys for NAbs. 23 Here, mjor concern is tht the ddition of empty prticles could increse the AAV cpsid lod, risking induction of cpsid dose-relted cytotoxic immune response nd perhps competing with full AAV prticles for effective trnsduction. 4 No stndrdiztion of processes leding to the genertion of empty prticles exists, nd it hs recently been reported tht prtilly empty cpsids (coproduced with vector) results in greter pprent liver inflmmtion thn when using genome-contining AAV vectors only. 42 With this bckdrop, we hve pplied the strtegy of directed evolution to develop novel AAV vrints in the presence of humn NAbs from AAV-treted ptients. 6,35,38,43 We successfully isolted severl AAV mutnts in the presence of ser from three ptients when selected in vitro. Some of these chimeric cpsids could escpe the NAb ctivity from ll three ptients, while others induced higher trnsduction in the presence of the cognte serum from which the evolutionry pressure ws derived. Such results suggest tht it is necessry to develop individul NAb escpe mutnts in the presence of serum from the specific subject, lthough it is possible (s we hve lso found) tht some of AAV mutnts isolted from one subject serum my confer generic NAb escpe bility cross other ser. Becuse the mechnism of vrying AAV trnsduction efficiencies in different cells nd tissues is uncler, nd it is well known tht the AAV trnsduction profile in vitro does not typiclly correlte in vivo, we hve lso determined tht it is impertive to develop tissue-tropic AAV vectors with the bility to escpe NAbs. Herein, we dvnced the in vitro pproch to in vivo selection in mouse muscle. Six mutnts were isolted from mouse muscle tht could evde serum NAbs from ptient smples. The dt clerly indicte the fesibility of developing NAb evsion mutnts with enhnced AAV trnsduction efficiency but more importntly provide rodmp for protein domins required for these functions in chimeric cpsids. Additionlly, the results obtined in this nd our studies strongly suggest tht selection of NAb escpe AAV mutnts should be performed in the presence of individul ntiserum but not using pooled ntiser (e.g., intrvenous immunglobin). Pooled ser re collected from different popultion of humn subjects. In this mixed ser, some subjects hve no AAV NAb nd others hve different titers of NAb (s high s :,000). 7 When ser from these subjects re pooled together, the titer of AAV NAb ctully is decresed due to dilution, nd the NAb ctivity from some subjects my dispper due Moleculr Therpy vol. 24 no. jn

10 The Americn Society of Gene & Cell Therpy to low NAb titer. Therefore mutnts isolted ginst pooled ntiser my only be pplied to subjects with high NAb titer. Structurl studies hve demonstrted tht there re nine vrible regions (VRs) on the AAV virion surfce tht re responsible for AAV serotype tropism nd trnsduction s well s NAb recognition For n individul subject, NAb ctivity will vry in strength to distinct serotypes. The cpsid genomes of our AAV shuffled librry re mixture of AAV serotypes through 9. Thus, Nb-binding sites from one but not nother serotype my be present on the chimeric virion surfce. Therefore, using the directed evolution/shuffling pproch we cn identify NAb binding domins by nlyzing the mutnt cpsids tht possess NAb escpe bilities. We first crried out phylogenetic nlysis of different isoltes obtined from both in vitro nd in vivo studies to ssess the diversity of mutnts obtined by directed evolution (Figure 6). Consistent with functionl dt, we observed tht the brod NAb evding clones, AAV00-, AAV002-4, nd AAV003-9, re distinct isoltes belonging to different cldes. It is interesting to note tht the brod NAb escping muscle isolte, Mus2, is phylogeneticlly similr to AAV00-, despite being recovered from skeletl muscle tissue in vivo. Furthermore, AAV002-4 is locted in the sme clde contining AAV/. Overll from different muscle isoltes, we noted tht every escpe mutnt contined -derived cpsid sequence t the C-terminus. Consistent with this observtion, hd superior bility to escpe NAbs thn ny other serotype in the presence of ptient 3 serum both in vitro nd in vivo. Findings bsed on swpping the C-terminus of nd further supported tht NAb binding sites from ptient 3 serum were loclized to the C-terminus of the (but not ) cpsid. Therefore, modifiction of the cpsid C-terminus cn evde NAb inhibition of AAV trnsduction. It is interesting to note tht Mus3 nd 2 were most successful t inducing muscle trnsduction fter direct injection mong the six mutnts with high muscle tropism; however, their NAb evsion bilities vried drmticlly: Mus3 ws ble to escpe its cognte serum NAb but not ser from the other two ptients, wheres Mus2 could only evde NAb ctivity of ser from other ptient ser but not from ptient 3. The sequence lignment between the two mutnts reveled sequences downstrem of residue 448, nd tht both cpsids hd sequences upstrem of residue 37 contining vrible point muttions. However, ech hd insertions from wide vriety of prentl serotypes locted between residues (Mus3 hs nd, Mus2 hs ). Other observed differences between the Mus3 nd 2 mutnts re locted in residues 327, 329, nd 33, which hve previously been shown to be locted ner the fivefold pore surfce, region tht hs not been identified s determinnt of ntigenic rectivity in previous studies. 47 It is well known tht AAV vectors from different serotypes disply vrious muscle tropisms: AAV or, which differ by six residues, induce the best muscle trnsduction in mice. 48,49 Previous studies hve shown tht chimeric vectors with residues 350 to 430 of VP induce much higher muscle trnsduction when substituted with the corresponding sequence from AAV. 50,5 Consistent with the chimeric vector study, 4 mutnts (Mus3,, 9, nd 24) contined residues 347 to 446 of VP (contining VRIII) derived from AAV/6, nd synthetic mutnts specificlly designed to swp corresponding sequences from led to higher muscle trnsduction. Importntly, the pproch used here not only identified chimeric mutnts with NAb evsion, but lso provided n effective pltform to identify motifs responsible for tissue tropism. Finlly, we crried out structurl modeling of ech representtive brod NAb escping mutnt to determine whether structure function correltion cn be estblished. Specificlly, we modeled trimers nd pentmers of different VP subunit sequences derived from the clones AAV00-, AAV002-4, nd AAV003-9 s well s the Mus2 isolte (Figure 6b). We noted tht the region most vried from the cpsid sequence is between residues 27 nd 36. This prominent surfce domin contins VR I nd hs been implicted in erlier ntigenicity studies. 3 Furthermore, it is interesting to note tht AAV00-, which belongs to the sme clde s Mus2 nd AAV003-9, lso contins the sme region, lbeit with fewer residues (233 35). These results suggest tht it is possible to identify single immunodominnt epitopes on the AAV cpsid tht cn be engineered to medite NAb evsion. In prticulr, VR I ppers prticulrly suitble for such pplictions without dversely ffecting trnsduction efficiency. The current study hs certin limittions. For instnce, we only selected AAV mutnts in muscle tissue in the presence of humn serum nd only generted 30 unique clones for sequencing, which my be due to the different muscle fibers trnsduced from AAV librry virus nd Ad. Six out of 8 vilble mutnt cpsids induced higher muscle trnsduction thn AAV serotypes, 2, 8, or 9, but ll were slightly reduced compred to the superior muscle trnsduction of. Interestingly, most mutnts induced higher trnsgene expression in muscle thn in the presence of ptient NAbs. These results strongly support the fesibility of selection of AAV NAb escpe mutnts in the presence of NAb. By performing dditionl rounds of selection in the presence of humn serum, perhps we could hve incresed the likelihood of isolting mutnt with both NAb evsion nd muscle trnsduction similr to. Another rguble limittion of this study is tht the mutnts recovered from muscle hd lower musculr trnsduction thn upon systemic dministrtion, lthough most displyed high muscle trnsduction fter i.m. injection. Most likely, these mutnts do not hve high vsculr permebility to trnsduce muscle s compred to. Mybe better strtegy to test in future studies would be to isolte AAV mutnts from muscles fter systemic dministrtion of the AAV librry nd humn serum s our results clerly demonstrte tht ech prmeter in the selection process gretly influences chimeric cpsid selection. In summry, we hve successfully recovered AAV mutnts in vitro nd from mouse muscle fter piring n AAV shuffled librry with NAb-contining humn serum. Although it is possible to obtin AAV mutnts tht evde NAb ctivity from different subjects, we found tht mutnts isolted from specific ptients re better t evding cognte serum-bsed NAbs thn NAbs from others. Most importntly, the isolted mutnts provide importnt informtion for identifying both NAb binding domins on the AAV virion surfce nd identifying unique motifs conferring muscle tissue tropism. These results estblish pltform to llow us to generte pnel of AAV mutnts isolted from different ptient ser, nd these mutnts will be tested for NAb evsion 62 vol. 24 no. jn. 206

11 The Americn Society of Gene & Cell Therpy cpcity in ptients involved in clinicl tril. If no mutnts re ble to escpe NAb from specific ptient, it is necessry to develop ptient-specific mutnts in the presence of cognte serum. MATERIALS AND METHODS Cell lines. HEK293 cells nd RC32 cells (HeL cells with stble rep expression) were mintined t 37 C in 5% CO 2 in Dulbecco s modified Egle s medium with 0% fetl bovine serum nd 0% penicillin streptomycin. Serum smples. Ser were collected from three DMD ptients in our phse clinicl tril t dy 00 fter i.m. injection of.5/mini-dystrophin vector nd stored t 80 C (ref. 6). AAV virus production. A three plsmid trnsfection method ws used to produce AAV vector, described previously. 52 Briefly, AAV trnsgene plsmid ptr/cba-luc, AAV helper plsmid, nd Ad helper plsmid pxx6-80 were cotrnsfected into HEK293 cells. Sixty hours posttrnsfection, HEK293 cells were collected nd lysed. Superntnt ws subjected to CsCl grdient ultr-centrifugtion. Frctions contining AAV were collected nd tittered by dot-blot. NAb escping AAV mutnts screen in vitro HEK293 cells were seeded in six-well plte t the time of the experiment prticles of AAV cpsid shuffling librry in 0 μl PBS ws incubted with 0 μl of undiluted ptient serum for 2 hours t 4 C. AAV/serum mixtures were pplied to cells with the ddition of Ad dl309 t multiplicity of infection (MOI) of 5. Forty-eight hours posttretment, cells were hrvested nd subjected to three freeze/thw cycles. Lystes were centrifuged t 2,000 rpm for 0 minutes to remove cell debris, nd superntnt ws used to repet the process for three more cycles to mplify virus in the bsence of ptient serum. After the fourth cycle, Hirt DNA ws extrcted from HEK293 cells, nd AAV mutnt cpsids were mplified by PCR with the following primers: F 5 -CAACTCCATCACTAGGGGTTC nd R 5 -CATGGGAAAGGTGCCAGA, which re loclized t the rep nd ITR, respectively. PCR using PfuUltr High-Fidelity DNA polymerse (Agilent Technologies, Snt Clr, CA) ws conducted with the following conditions: 94 C 30 seconds, 53 C 30 seconds, 72 C 3 minutes for 35 cycles. PCR products were digested with SwI nd XbI nd ligted into pxr2 digested with the sme endonucleses. Clones were sequenced by the UNC-CH Genome Anlysis Fcility, nd cpsid sequence lignment ws nlyzed with VectorNTI (Invitrogen, Grnd Islnd, NY). Hirt DNA purifiction. Low-moleculr-weight DNA ws extrcted from HEK293 cells s described previously. 53 Briefly, HEK293 cell pellets were resuspended in 728 μl of Hirt buffer (20 mmol/l Tris HCl, 20 mmol/l EDTA, ph 8.0) nd lysed with the ddition of 46 μl of 0% sodium dodecyl sulfte. 28 μl of 5 mol/l NCl ws dded to the cell lyste solution nd incubted for hour on ice. Lyste ws centrifuged t 5K rpm t 4 C for 30 minutes. Superntnt ws collected, nd DNA ws extrcted with phenol/chloroform. DNA ws precipitted with isoproprnol, nd resuspended in 50 μl TE buffer (0 mmol/l Tris HCl, mmol/l EDTA, ph 8.0) contining 00 μg/ml of DNse-free RNse. In vitro trnsduction nlysis cells were seeded in 48-well plte for 2 hours. 0 8 prticles of AAV/luc ws dded. Forty-eight hours postinfection, cells were lysed using Pssive Lysis Buffer (Promeg, Durhm, NC). Cell lyste ws trnsferred to 96-well plte to mesure luciferse ctivity with Wllc420 Victor2 microplte reder. For inhibition ssy of trnsgene expression in the presence of serum, 0 8 prticles of AAV/luc in 0 μl PBS were incubted with 0 μl serum for 2 hours t 4 C, then dded to HEK293 cells. Luciferse ctivity ws mesured 24 hours post-aav infection. NAb nlyses. NAb nlyses were performed s described previously with slight modifiction. 7,27 Ser were serilly diluted twofold with PBS. RC32 cells were seeded in 300 μl cell medi in 48-well plte for 3 to 4 hours. 0 8 prticles of AAV/luc ws incubted with ech serum dilution in PBS for 2 hours t 4 C in totl volume of 25 μl. AAV/ser mixtures were dded to cells in finl volume of 25 μl, which contined prticles of Ad dl309. Cells with AAV/ser mixtures were incubted for 24 hours t 37 C. NAb titers were defined s the highest dilution for which luciferse ctivity ws 50% less thn controls (no ser). NAb escping AAV mutnts isolted from mouse muscles. 0 0 prticles of the AAV cpsid shuffling librry virus in 50 μl PBS were mixed with 50 μl undiluted serum from ptient 3 for 2 hours t 4 C. The AAV/serum mixture ws delivered vi i.m. injection into the hind leg muscle of 6-week-old femle BALB/c mice. Three dys posttretment, Ad virus dl309 ws delivered vi i.m. injection into the sme muscle t MOI of 0 7 to mplify AAV genomes in vivo (Supplementry Figure S5). Muscle ws collected 2 dys post Ad dministrtion, nd totl DNA ws extrcted using Qigen Kit (Qigen, Vlenci, CA). PCR ws crried out for mplifiction of AAV mutnt cpsids, s done bove, using muscle DNA s templte. PCR products were cloned into pxr2 bckbone. Colonies were sequenced nd used to generte AAV/ luc mutnt vectors. Housing nd hndling of mice ws crried out in complince with the Ntionl Institutes of Helth guidelines nd n Institutionl Animl Cre nd Use Committee pproved protocol t UNC-CH. Chrcteriztion of AAV mutnts in mice. Six-week-old femle BALB/c mice received 0 prticles of AAV/luc vi retro-orbitl injection. Luciferse expression ws imged week postinjection using Xenogen IVIS Lumin (Cliper Lifesciences, Wlthm, MA) following i.p. injection of d-luciferin substrte t 20 mg/kg (Nnolight Pinetop, AZ). Bioluminescent imges were nlyzed using Living Imge (PerkinElmer, Wlthm, MA). For muscle trnsduction, 0 9 prticles of AAV/luc were injected into the gstrocnemius of 6-week-old femle BALB/c mice. Mice were imged t the indicted time points. Quntittion of luciferse expression in tissues. Animls utilized for imging studies were scrificed 2 weeks post AAV injection, nd the following orgns were collected: liver, spleen, kidney, hert, lung, skeletl muscle (gstrocnemius), nd brin. Tissue ws minced nd homogenized in pssive lysis buffer. Tissue lystes were centrifuged t 0K rpm for 5 minutes to remove cellulr debris. Superntnt ws trnsferred to 96-well pltes for luciferse ctivity nlysis s described bove. Totl protein concentrtion in tissue lystes were mesured using the Brdford ssy (BioRd Lbortories, Phildelphi, PA). AAV mutnt cpsid cloning. AAV cpsid swp cssettes were generted by PCR with primers listed in Supplementry Tble S. PCR products were ligted into the pxr2 bckbone. To generte the pxr-262 clone, we first mde the construct pxr-62. To generte pxr-62, we obtined PCR product with primers F/R2-262 using pxr6 s templte nd PCR product with primers F3-262/R using pxr2 s templte. Both PCR products were digested with SlI nd NotI nd ligted into pxr2 digested with the sme endonucleses. Next, to generte pxr-262, we obtined PCR product with primers F2-262/R using pxr-62 s templte nd PCR product with primers F/R-262 using pxr2 s templte. Both PCR products were digested with SlI nd NotI nd ligted into pxr2 digested with the sme endonucleses. To generte clone pxr-2/8, we obtined PCR product with primers F/R-2/8 using pxr2 s templte nd PCR product with primers F2-2/8/R using pxr8 s templte. Both PCR products were digested with SlI nd NotI nd ligted into pxr2 digested with the sme endonucleses. To generte clone pxr-8/2, we obtined PCR products with primers F3-262/R using pxr2 s templte nd PCR product using F/R2-2/8 using pxr8 s templte. Both PCR products were digested with SlI nd NotI nd ligted into pxr2 digested with the sme endonucleses. Colonies were verified by DNA sequencing. Moleculr modeling. Structurl homology models of the AAV cpsid mutnts were obtined using the SWISS-MODEL online server ( swissmodel.expsy.org/), 54 with the crystl structure of VP3 (PDB Moleculr Therpy vol. 24 no. jn