© The American Society of Gene & Cell Therapy original article
`
`See page 249
`
`Phase 1 Gene Therapy for Duchenne Muscular
`Dystrophy Using a Translational Optimized AAV
`Vector
`
`DawnE Bowles", Scott W] McPhee’, Chengwen Li?, Steven J Gray?, Jade | Samulski?,
`Angelique S Camp%, Juan Li’, Bing Wang>, Paul E Monahan?, Joseph E Rabinowitz®, Joshua C Grieger*,
`Lakshmanan Govindasamy’, Mavis Agbandje-McKenna’, Xiao Xiao’ and R Jude Samulski?
`
`‘Departmentof Surgery, Division of Surgical Sciences, Duke University Medical Center, Durham, North Carolina, USA; *Asklepios BioPharmaceuticalInc.,
`ChapelHill, North Carolina, USA; ?3Gene Therapy Center, University of North Carolina at ChapelHill, ChapelHill, North Carolina, USA;*Joint Vector Core,
`University of North Carolina, ChapelHill, North Carolina, USA; ‘Eshelman Schoolof Pharmacy, University of North Carolina, ChapelHill, North Carolina,
`USA; ®Center for Translational Medicine, Department of Medicine, ThomasJefferson University Philadelphia, Philadelphia, Pennsylvania, USA;
`’Departmentof Biochemistry and MolecularBiology, Center for Structural Biology, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA
`
`Efficient and widespread genetransfer is required for
`successful treatment of Duchenne muscular dystrophy
`(DMD). Here, we performed the first clinical trial using
`a chimeric adeno-associated virus (AAV) capsid variant
`(designated AAV2.5) derived from a rational design strat-
`egy. AAV2.5 was generated from the AAV2 capsid with
`five mutations from AAV1. The novel chimeric vector
`combines the improved muscle transduction capacity
`of AAV1 with reduced antigenic crossreactivity against
`both parental serotypes, while keeping the AAV2 recep-
`tor binding.
`In a randomized double-blind placebo-
`controlled phase | clinical study in DMD boys, AAV2.5
`vector was injected into the bicep muscle in one arm,
`with saline control in the contralateral arm. A subset of
`patients received AAV empty capsid instead of saline
`in an effort to distinguish an immune response to vec-
`tor versus minidystrophin transgene. Recombinant AAV
`genomes weredetectedin all patients with up to 2.56
`vector copies per diploid genome. There wasnocellular
`immuneresponse to AAV2.5 capsid. This trial established
`that rationally designed AAV2.5 vector wassafe and well
`tolerated, lays the foundation of customizing AAV vectors
`that best suit the clinical objective (e.g.,
`limb infusion
`gene delivery) and should usher in the next generation
`of viral delivery systems for human gene transfer.
`Received 17 June 2011; accepted 5 October 2011; published online
`8 November 2011. doi:10.1038/mt.2011.237
`
`inherited, caused by mutations in dystrophin, a large (427kDa)
`cytoskeletal protein that is normally expressed in skeletal and car-
`diac muscle, as well as smooth muscle, brain, andretina in various
`isoforms.' The incidence of DMD is 1 in 3,500-5,000 newborn
`males.” The gene encoding dystrophin is the largest identified to
`date,' and the risk of spontaneous mutationis high (1/10,000 germ
`cells).
`Because DMDis caused by recessive and monogenic muta-
`tionsin the dystrophin gene, this disease is thought to be amenable
`to correction or improvementby gene therapy. Gene replacement
`therapy for DMD is a promising strategy because in theory it
`could benefit all DMDpatients regardless of the natureoftheir
`genetic mutations e.g. deletions and point mutations. A number
`of additional drugs are in development and include alternative
`gene therapy approaches,cell therapy, antisense oligonucleotides,
`and small molecule drug therapies.? However, DMDpresents with
`a multitude of challenges for the development ofeffective treat-
`ments including the largest disease gene that requires miniaturi-
`zation to be compatible with gene replacementvectors, the very
`large mass of target tissue that is widely distributed throughout
`the body with layers of biological barriers, and unknown changes
`at the cell membranelevel that may impact on vector binding and
`uptake. Furthermore, the immunological components of DMD
`pathophysiology may also impact on the development anddeliv-
`ery of novel therapeutics and therefore assays of immune system
`recognition and response mustbe carefully integrated into clinical
`trial designs.
`Recombinant adeno-associated virus (rAAV) vectors car-
`rying a miniaturized functional dystrophin gene (designated
`minidystrophin) have the potential to arrest or reverse muscle
`failure.*° Comprehensive proof of concept andpreclinical testing
`has evaluated the effectiveness of AAV-minidystrophin gene
`delivery in animal models of DMD including the mdx mice and
`dystrophin/utrophin double knockout mice. Minidystrophin
`
`
`INTRODUCTION
`
`Duchenne muscular dystrophy (DMD) is the most common
`severe, life-threatening form of muscular dystrophy in child-
`hood. DMD is associated with progressive muscle degeneration,
`weakness, and mortality. DMD is X-linked and is genetically
`
`Thefirst three authors contributed equally to this work.
`Correspondence: R Jude Samulski, Gene Therapy Center, University of North Carolina at ChapelHill, 7119 Thurston Bowles CB 7352 ChapelHill,
`North Carolina 27599-7352, USA. E-mail: ris@med.unc.edu or Xiao Xiao, University of North Carolina at ChapelHill, Eshelman School of Pharmacy,
`CB # 7362, Genetic Medicine Building, Chapel Hill, North Carolina 27599-7362, USA. E-mail: xxiao@email.unc.edu
`
`Molecular Therapy vol. 20 no. 2, 443-455feb. 2012
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`DEPOSITION
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`Figure 1 Aminoacid candidates responsible for efficient skeletal muscle transduction. (a) Capsid amino acids of low skeletal muscle transduc-
`ing serotypes (AAV2, AAV3) versus high skeletal muscle transducers (AAV1, AAV6, AAV7, AAV8, AAV9) were aligned using the Vector NTI program
`(Invitrogen). Alignments were examined for distinct amino acids of AAV2 from the others. See text for additional modeling criteria. Amino acids
`boxed or marked with arrows were deemedto be ofinterest. AAV2.5 is composed of thefive amino acids indicated by * and @.(b) Location of the
`five amino acids on a single VP subunit which were modified in the AAV2.5 variant. Notice that the five amino acids are located on opposite posi-
`tions of one subunit. (¢) Location of the same five amino acids(circles and arrows) in the context of an assembled AAV capsid pentamer. Notice that
`the five amino acids are now in close proximity when two subunits are assembled. The five amino acid changesare located near the twofold axis of
`symmetry. AAV, adeno-associatedvirus.
`
`expressed after AAV delivery to dystrophin deficient models has
`been shownto:correctly localize to the sarcolemma,restore the
`missing dystrophin-associated protein complex to the cell mem-
`brane, ameliorate dystrophic pathology in mdx muscle, normalize
`myofiber morphology, normalize cell membraneintegrity, restore
`missing dystrobrevin complex, partially restore a-syntrophin
`association with the cell membrane,partially restore nitric oxide
`synthase activity, reduce musclefibrosis, reduce myofibercentral
`nucleation,
`improve whole-body endurance and muscle force
`transduction, reduce kyphosis and limb deformation, andincrease
`generalhealth andlifespan.**
`Genetic strategies that target the muscular dystrophies will
`ultimately require widespread delivery to a large volume of
`skeletal musculature and/orcardiactissue. Strategies to improve
`transgene expression to the musculature have included the use
`of AAV serotypes other than AAV2andefforts to evolvetissue
`specificity variants by directed capsid evolution as well as mosaic
`vector with a mixture of capsid from different serotypes.*"? It
`is clear that no single natural AAV serotype will be useful for
`
`every clinical application, nor will directed evolution evolveall
`characteristics desirable for a clinical scenario simultaneously.
`Any given serotype may contain biological characteristics both
`beneficial and detrimental
`to the given clinical application.
`Instead of attempting to “fit” a known AAVserotypeto a disease
`process or coevolve multiple traits in a single capsid, we chose
`to use a rational approach to identify capsid regions on alter-
`native AAV serotypes responsible for enhanced skeletal muscle
`transduction and to combine these modifications into the AAV2
`
`capsid which offers the benefits of a well-defined safety profile
`coupled with purification ease. The availability of capsid protein
`sequencesfrom several AAV serotypes, muscle transduction pro-
`files with different serotypes of AAV vector and antigenic epitope
`information for AAV2, combined with the three-dimensional
`structure of the AAV2 capsid! provided us valuable information
`to rationally design efficient vectors for clinical trials. Through
`mutagenesis with insertion and substitution, a chimeric AAV2-
`AAV1 vector, dubbed AAV2.5, was designed to contain desir-
`able biological properties from both parent viruses. Compared
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`to AAV2, AAV2.5 hassimilar transduction efficiency in several
`cell lines and binds to heparin sulfate in vitro. However, AAV2.5
`induces stronger transductionin skeletal muscles than AAV2 and
`demonstrated lower crossreactivity to AAV2 neutralizing anti-
`bodies (Nab).
`The novel AAV2.5 capsid offering improved skeletal muscle
`gene transfer efficiency and potentially reduced immunogenicity
`compared with naturally occurring serotypes was next evaluated
`ina clinicaltrial for DMD utilizing the minidystrophin transgene
`cassette. The initial development of AAV2.5-minidystrophin
`clinicaltrial capitalized on the fact that AAV2 wasthe only sero-
`type approved for clinical use, and AAV1 wasthe only other
`AAV serotype underserious consideration for clinical studies.
`To this end, a subset of amino acids (5aa) in AAV type 1 were
`constructed into type 2 capsid backbone. The engineering and
`testing of this chimeric capsid in clinical setting has provided
`a paradigm where as the investigator is no longer obligated to
`naturalviral isolates for gene delivery and cantherefore address
`additional clinical concerns (e.g., capsid immuneresponse) that
`lie outside of primary objective of measuring therapeutic trans-
`gene expression.
`Therefore the novelnature of the proposed therapeutic strategy
`and study population led to the inclusionof careful monitoring of
`both humoral andcell-mediated immuneresponsesin this phase I
`clinical trial. We have recently reported elsewhere on unexpected
`findings related to dystrophin immunity observedin thistrial.’
`A subsetofpatients were shownpost-hoc to have had pre-existing
`immunity to dystrophinepitopes believed to be expressedby rever-
`tant myofibers, and cellular immuneresponses to minidystrophin
`epitopes werealso observed. Wedetail herein the immuneresponse
`to the novel AAV2.5 capsid as well as other study endpoints, such
`as: (i) successful vector transgene delivery to all patients at each
`dose based on PCRanalysis of biopsy sectioning,(ii) no difference
`in immuneinfiltration when comparing placeboto vectortreated
`arms,(iii) lack of detectable immuneresponse in empty vector only
`tissues, (iv) no CTL response to chimeric capsid at any dose.All
`andall, this trial established that rationally designed AAV capsid
`wassafe, well tolerated and lays a foundation of customizing AAV
`vectors that ideally suit the clinical objective(e.g., heart tropic,liver
`detargeted, neuroselective,etc.).
`
`RESULTS
`Rational design of AAV chimeric vectors
`Several of the AAV serotypes characterized to date trans-
`duce mouse skeletal muscle with greater efficiency than AAV2,
`e.g., AAVI, AAV7, AAV8, and AAV9."*-!7 In aneffort to identify
`
`Table 1 The characteristics of chimeric variants
`
`Clinical Evaluation of Custom-designed AAV Capsid in DMD Patients
`
`candidate amino acids responsible for enhanced transduction of
`skeletal muscle the VP1 amino acid sequencesofthese serotypes
`with high muscular tropism were compared to AAV2 using an
`alignment (Figure la). The criteria for selection of amino acid
`candidatesare: (i) they must differ from AAV2 andbesimilar to
`the high muscle tropism serotypes, (ii) they must be located in
`a structurally variable region (VR) on the capsid surface,'*” or,
`(iii) they must be located in an AAV2antigenic region thatis rec-
`ognized by an antibody. Shown boxedin Figure 1a are the amino
`acids which metall criteria with the exception of one aminoacid
`depicted by @ (residue 716, AAV2 Vp1 numbering) duetoits
`close proximity to four other aminoacids.
`Three variants were initially generated in which AAV2 was
`modified to resemble AAV1: AAV2.5, AAV2-Q325T/T329V, and
`AAV2-T450N/Q457N.In the AAV2.5 variant four residues were
`substituted with AAV1 amino acids (Q263A, N705A, V708A,
`T716N, AAV2 numbering) and one AAVI1 amino acid (T265,
`AAV1 numbering) was inserted into the AAV2 capsid (amino
`acids indicated by the asterisks (*) and @ in Figure la and the
`3D model shownin Figure 1b,c). These mutations areall on the
`VRs of the virion surface (VR I and VR IX,Figure 1b). Based
`on the AAV atomic structure, AAV2.5 was designed becauseresi-
`dues 263 and 265 intercommunicate with residues 706, 708 and
`717 (Figure Ic). The locationsofall five amino acid mutations in
`AAV2.5 are close to the A20 antigenic epitope.'*”"! The AAV2-
`Q325T/T329V variant (AAV2 numbering; depicted by arrows on
`Figure la)) partially met the set criteria, the amino acids are con-
`servedin AAV8 butdiffer in AAV1, AAV7, and AAV9,and are not
`close to a mapped AAV2antigenicsite. The AAV2-T450N/Q457N
`(boxed only in Figure 1a) contains aminoacids located in VR V.
`The amino acid mutations in the three AAV2 variants did
`not influence the ability to generate recombinant AAV vectors
`compared to unmodified AAV1 and AAV2 capsids containing
`the same transgene cassette as judged by physical particle titers
`(Table 1). The AAV2.5 variant resembled AAV2 with respect to
`its ability to bind heparinin affinity columnsusedfor purification
`(Table 1). Furthermore, the transduction profiles of these variants
`in cultured HeLa, Cos, and 293 cells were morelike that of AAV2
`than AAV1 (Table 1).
`
`Genetic modification of the AAV2 capsid can improve
`muscle transduction
`
`in vivo
`To determine whether amino acids responsible for
`transduction could be accurately predicted, the variants with a pack-
`aged luciferase gene (AAV2.5-luciferase, AAV2-Q325T/T329V-
`luciferase, and AAV2-T450N/Q457N) were evaluated for their
`
`Virus
`
`Parental AAV2
`
`Physical titer
`(vg/pl)
`1.3-8.5 x 108
`
`Heparin
`binding
`++
`
`Heparin
`competition
`++
`
`Muscle
`A20 neutralizing
`
`293
`Hela
`Cos1
`transduction in vivo
`activity
`+444
`+t+++
`+444
`+
`+++
`
`=
`HH
`+
`+
`+
`=
`=
`13-15 x 10°
`SerotyPe aAV
`-
`+++
`+4+4++
`+444
`+4+++
`++
`++
`5.0-9.2 x 108
`AAV2.5
`Q425T/T329V
`6.9-13 x 108
`N/D
`N/D
`+444
`N/D
`+444
`+
`N/D
`
`T450N/Q457N
`4.4-9.3 x 108
`N/D
`N/D
`+4+4++
`N/D
`+++
`++
`N/D
`Abbreviations: AAV, adeno-associated virus; N/D, not done; vg, vector genome.
`
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`Minimalcrossreactive neutralizing antibodies exist between
`AAV2andotherserotypes such as AAV1”(C. Liand RJ. Samulski,
`unpublished results). To determine whether the neutralizing
`antibodyprofile of AAV2.5 was more like AAV2 or AAVI, sera
`from mice treated with AAV1-luciferase, AAV2-luciferase, or
`AAV2.5-luciferase vectors were analyzed for neutralizing anti-
`bodycrossreactivity. Shown in Figure 3a are the dilutions of the
`sera from AAV2, AAV2.5, and AAV] injected mice needed for
`50% inhibition of infectivity of AAV2, AAV1, or AAV2.5 in 293
`cells. The sera from AAV2-treated mice neutralized infectivity of
`AAV2 moreefficiently than the infectivity of AAV2.5 byfivefold.
`A similar finding was observed in the examinationofthe ability
`of AAV1 serato inhibit the infectivity of AAV1 and AAV2.5. The
`converse was found to be true for sera from AAV2.5 injected
`animals which was observed to be four- and eightfold more
`efficient at neutralizing AAV2.5 compared to AAV2 or AAV1,
`respectively. Therefore AAV2.5 with minimal changeof 5 aa has
`antigenic properties that are distinct from those of the paren-
`tal viruses suggesting that the engineered AAV2.5 capsid may
`eliminate the AAV2 or AAV1 antibody recognized epitopes or
`changevirion three-dimension structure and suchthatit is less
`
`a 1,200
`
`S 800
`=
`8Zz
`
`400
`
`AAV2
`m AAV
`® AAV2.5
`
`
`b
`
`
`
`AAV2
`AAV1
`AAV2.5
`
`Sera from mice
`@=1.00x10°
`
`ability to transduce skeletal muscle following injection of equiva-
`lent genome-containingparticles into the gastrocnemius muscle of
`BALB/c mice. Luciferase expression was evaluated over time using
`biophotonicin vivo imaging and comparedto the parental AAV1 and
`AAV2(for AAV2.5) or AAV2 (for AAV2-Q325T/T329V) viruses
`(Figure 2a andb,respectively). In skeletal muscle, the AAV2.5 vari-
`ant consistently produced higher transgene expression than AAV2
`at all time points tested albeit not to the identical level as observed
`with AAV1 (Figure 2a). AAV1 exhibited 5-12.5-fold higher levels
`of light emission than AAV2; whereas AAV2.5 exhibited 1.8-5.5-
`fold higher light emission than AAV2. Mice injected with AAV2.5
`exhibited high levels of expression up to 8.4 monthspostinjection.
`Theexpressionlevel of the AAV2-Q325T/T329Vvariant was indis-
`tinguishable from that ofAAV2 (Table 1 and Figure 2b) and was not
`tested further. The third variant, AAV2-T450N/Q457Nexhibited a
`3.5-fold enhancementin transgene expression over AAV2(‘Table 1),
`but only at a later time point postinjection (day 42 postinjection)
`and wasnottested anyfurther.
`
`Unique antigenic properties of the AAV2.5 capsid
`Theantigenic profile of the AAV2.5 vector was exploredfirst by
`examiningits recognition by the well characterized A20 anti-AAV2
`monoclonal antibody.”! These studies revealed that the ability of
`the A20 antibodyto recognize the AAV2.5 vector is extinguished
`comparedto its strong recognition of AAV2 (Table 1). Thus this
`data suggest that AAV2.5 may have an immuneprofile distinct
`from AAV2.
`
`1.00x10”
`
`1.00x10°
`
`1.00x10°
`
`1.00x10* RLU/RPI
`
`b
`
`600x10° y————
`|
`500x10° +-{—4—pave to400x108 4-4 i" 325 329}_. _
`[
`300x10°
`
`|
` 200x10°
`
`100x10° |
`
`00x10° |
`
`Dayspostinjection
`
`Figure 2 Evaluation of skeletal muscle transduction of AAV2 mutants.
`(a) Skeletal muscle transduction of AAV1, AAV2, and AAV2.5 examined
`over time (days 3, 7, 21, 28, 42) using in vivo biophotonic imaging.
`Therelative light units per region of interest in each injected mouse (n=
`6) are graphed over time. (b) Graphical representation of quantity of
`emitted light from transduction of AAV2 and AAV2-Q325T/T329V. The
`relative light units per region of interest in each injected mouse leg (n =
`6) are graphed overtime. AAV, adeno-associatedvirus.
`
`AAV2
`m AAV2.5
`
`40-
`
`|
`
`&S
`
`30
`s
`8@ 20
`a
`
`‘
`
`E
`
`d
`
`100-
`
`Ex
`
`0+ a n=
`No
`2-<10
`10-<100
`Nabtiter
`
`Figure 3 Neutralizing antibody analysis to AAV2.5.(a) Crossreactive
`Nab between AAV1, AAV2, and AAV2.5. C57 mice were immunized with
`1 x 10!° particles of AAV/luc vectors via muscular injection. Thirty days
`later, sera from three mice wascollected for Nab analysis. (b) The effect
`of AAV2 Nab on AAV2.5-induced transgene expression in vivo, Mice were
`immunized with AAV2/AATviruses(left three panels) or not immunized
`(right panel), 2 months later, AAV2.5/luciferase (mouse right leg) and
`AAV2/luciferase (left leg) vectors with different dosages were applied in
`the same mice intramuscularly (5 x 10° particles,
`1 x 10'° particles, or
`5 x 10"? particles), imaging was taken 6 weekslater post-luciferase vector
`injection. (¢) Neutralizing antibody assay for human sera. The sera from
`36 human subjects were detected for Nab against AAV2 and AAV2.5.
`AAV, adeno-associated virus.
`
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`likely to be neutralized by the sera of animals pretreated with
`AAV2 and AAVI.
`
`Genetic modification of the AAV2 capsid enables
`repeat administration of variant vectors
`Theserological data described above suggested that AAV2.5 was
`antigenically distinct from the parental viruses. To test whether
`this phenotype would makethis variant refractory to pre-exist-
`ing antibodies and allow readministration in subjects previously
`treated with AAV2 vectors, in vivo studies were performed in
`which mice previously injected with an AAV2-AATvector were
`subsequently injected intramuscularly (i.m.) with increasing
`doses of either AAV2-luciferase or AAV2.5-luciferase vectors.
`
`For direct comparison, each mouse received the same dose ofthe
`two vectors in separate leg muscles. In vivo biophotonic imag-
`ing performed at 6 weeks postinjection ofthe luciferase express-
`ing virus revealed no detectable expression from either vector
`at low doses (5 x 10° particles, Figure 3b). However,at higher
`doses (1 x 10" particles (Figure 3b) and 5 x 10" particles per
`leg (Figure 3b)) the AAV2.5-luciferase administered legs con-
`sistently exhibited elevated transgene expression (~10-20-fold)
`over its AAV2-luciferase injected counterpart (Figure 3b). The
`enhanced transduction by AAV2.5 is interpreted as being due
`to both its inherently higher skeletal muscle transduction com-
`pared to AAV2,as evidencedbyits ~2-5.5-fold higher transgene
`expression over AAV2observedin control mice with no previous
`exposure to AAV2 (Figures 2 and 3b, right panel)as wellastoits
`ability to overcome pre-existing anti-AAV2neutralizing antibod-
`ies. However, although it is clear that the high dose of AAV2.5
`vector could escape AAV2 neutralizing antibody partially in vivo,
`we could notrule out the possibility that AAV2 neutralizing anti-
`bodies completely block AAV2.5 transduction after muscular
`injection in AAV2 pretreated mice when a much lowerdose of
`AAV2.5 wasused.
`
`Summaryoflate preclinical studies
`Sera from 36 individuals were screened for neutralizing antibodies
`and pre-existing neutralizing antibodytiters of >1:2 to AAV2 were
`seen in 75% of individuals while titers of >1:2 to AAV2.5 were
`
`seen in only 56% ofindividuals (Figure 3c). Of those individu-
`als that have AAV2 Nab, a very high Nab antibodytiter (2100)is
`found in 25% of these individuals; whereas, only 19.5 % of indi-
`viduals with Nab to AAV2.5 exhibit high Nabtiter. Generally the
`titer against AAV2.5 is 2-20-fold lowerthan that against AAV2 in
`the Nab positive population. The data from this humansera neu-
`tralizing antibody assay again support the conclusion that AAV2
`and AAV2.5 have different immuneprofiles.
`A penultimate preclinical study was conducted to assess possi-
`ble adverse interactions between the commonly prescribed corti-
`costeroid prednisone and AAV-minidystrophin in C57/BL10 mice.
`No discernable adverse interaction and influences on transgene
`expression were observed (data not shown, see Supplementary
`Materials and Methods—Preclinical AAV2.5-Minidystrophin
`Studies).
`The AAV-minidystrophin expression cassette including
`the cytomegalovirus (CMV) promoter and the polyadenyla-
`tion signal site in the vector plasmid was fully sequenced on
`
`Molecular Therapy vol. 20 no. 2 feb. 2012
`
`Clinical Evaluation of Custom-designed AAV Capsid in DMD Patients
`
`both strands except the inverted terminal repeats, which were
`difficult to sequence. A pivotal good-laboratory-practice toxic-
`ity and biodistribution study evaluated AAV-minidystrophin
`delivery in C57/BL10 mice with timepoints up to 36 weeks post
`vector administration and doses up to 1 x 10" vector genomes/
`kg, which was equivalent to 10 times the highest dose proposed
`in the clinical trial. Overall, the AAV-minidystrophin vector
`caused nosignificant toxicity in C57BL mice, with all animals
`surviving until scheduled sacrifice. Effects on clinical chemistry
`parameters in the toxicity study were within the normal refer-
`ence ranges, and there were nodifferential effects observed by
`histopathological analyses. There were no related effects on clini-
`cal hematology parameters or gross necropsy observations. PCR
`biodistribution evaluations indicated that the vector remained
`
`concentrated in the injected muscle (data not shown, see
`Supplementary Materials and Methods—Preclinical AAV2.5-
`Minidystrophin Studies).
`
`Regulatory approval process
`Theclinical protocol and Appendix M documents were reviewed
`by the Recombinant DNA Advisory Committee under the aus-
`pices of the Office of Biotechnology Activities at the National
`Institutes of Health (NIH). The Nationwide Children’s Hospital
`institutional
`review board approved the study protocol. A
`Pre-IND discussion was held with FDA beforeinitiation of the
`
`pivotal toxicology and biodistribution study, and upon conclu-
`sion ofpreclinical studies the IND was submitted to CBER/FDA
`(BB-IND 12936). The trial is registered with ClinicalTrials.gov
`(Reference no. NCT00428935).
`
`Clinical study design
`The experimental design was a randomized double-blind placebo-
`controlled study where vector was administered into one bicep
`andsaline control in the contralateral arm. In a subsetof patients,
`we substituted AAV empty capsid for saline in aneffort to distin-
`guish an immuneresponseto vector versus minidystrophin trans-
`gene. The active phase of the study included a 2-week baseline
`screening period,a 2-day inpatient periodfor vector injection and
`acute toxicity monitoring, and a 2-year outpatient follow-up and
`toxicity-monitoring period. Ongoing long-term follow-up will
`continue out to 15 years post vector injection. (Supplementary
`Figure $1). Blood and urine analyses were conducted in conjunc-
`tion with outpatient clinics on post administration days 8, 15, 30,
`43, 53, 60, 90, and 120 and months6,9, 12, 18, and 24.
`The study enrolled six DMD boys ranging in age from 5
`to 11 years and ranging in mass from 16 to 57kg, each with
`unique and defined dystrophin mutations. Four boys had been
`on corticosteroid medication schedules before vector injection
`(Table 2). All six patients were administered methylprednisolone
`(2mg/kg, limited to <1 g total) four hours prior to vector admin-
`istration, with repeat doses on the following two mornings. A
`MyoJect hypodermic needle (Oxford Instruments, Hawthorne,
`NY)wasusedto deliver 1.2 mlofvectorin three equivalent boluses
`spaced 0.5cm apart along an injection tract that was placed in a
`longitudinaltrajectory relative to the biceps muscle orientation.
`Administration of AAV2.5-Minidystrophin to each subject was
`staggered to allow for at least 6 weeks of follow-up before the
`
`447
`
`Sarepta Exhibit 1062, page 5
`
`Sarepta Exhibit 1062, page 5
`
`

`

`Clinical Evaluation ofCustom-designed AAV Capsid in DMDPatients
`
`Quantitative PCR of the vector genome in human
`next subject receiving the vector. There were two-dose cohorts of
`muscle biopsies
`three subjects each that received unilateral im. delivery of either
`The presence of the vector genomes wasassessed in muscle biop-
`6 x 10" vector genomesor 3 x 10" vector genomes. Dosing was
`sies from theleft and right arms 1.5 and 3 monthsafter injection.
`defined by locally administered dose as the vector was mediat-
`The transgene DNA wasdetected in 6 outof6 patients, in only one
`ing productionof a structural protein and notasecreted protein,
`and vector genomes remainedlocalized to the region ofthe injec-
`arm with nodetectable spread to the contralateral arm. The high-
`tion site. Internalized AAV capsid peptide competition for MHC
`est vector genome copy numberpernucleus (diploidcell genome)
`detected in each patient is shown in Table 2.
`presentation would also be more heavily influenced bythe local-
`ized dosing. Asa result of the inclusion of an empty capsid control
`Detection of minidystrophin protein in muscle tissue
`vector administeredto the last two subjects, there was a full log
`biopsy
`difference in the capsid dose between subjects 1-3 and5and 6.
`Cryothin-sectionsofthe muscle biopsies were immunofluorescently
`Biopsies were taken at 1.5 month in the majority of patients and
`stained with antibodies recognizing the dystrophin N-terminal
`out to 3 monthsin twopatients (Table 2).
`region (presentin both minidystrophin and endogenousrevertant
`myofibers) or the C-terminal region (absent in minidystrophin but
`present in revertant myofibers). Limited minidystrophin expression
`was observed with appropriate localization to the sarcolemma in
`two ofsix subjects. In patients 3 and6, only a few minidystrophin
`positive myofibers were detected by anti-N-terminusantibody but
`stained negative by anti-C-terminus antibody. However,in patients
`1, 2, 4 and 5, no minidystrophin positive myofibers were detected,
`suggesting that transgene expression waseither very pooror extin-
`guished in those patients (detailed in ref. 12).
`
`Clinical observations
`Physical examinations were unremarkable with no symptomsof
`fever, lymphadenopathy, organomegaly, and nosigns of inflam-
`mation at the injection site. During the 2-year-long active phase
`of the trial monitoring, no serious or mild adverse events have
`been observed in any subject. A few minor adverse events were
`observed including sore throats, rashes and nausea,but they were
`not considered related to the vector administration as they are
`commonly seen in this age group of subjects (detailed in ref. 12).
`Hematology and chemistry panels that included an assessment of
`liver function also indicated that the gene vector was well toler-
`ated in all subjects (Supplementary Figure $2). There were no
`abnormalelevations in the levels of creatine kinase, alkaline phos-
`phataselevels, or lymphocyte countsin all blood samplestested.
`
`AAV2.5-mediated gene delivery in patients
`Biopsies were undertaken in four subjects (subjects 1, 3, 4, and 6) at
`d43 post administration, and in two subjects (subjects 2 and 5)at
`d90 post administration. The biopsy procedureutilized ultrasound
`imaging to guide theretrieval of the injected tissue. A battery of
`assays were undertakenon the biopsied muscletissue and included:
`PCRanalysis of vector genomes, immunolabeling of minidystro-
`phin,tissue histochemistry including CD8 T-cell infiltration.
`
`Immunological studies
`Post administration hematological testing indicated no abnormal
`changes in white blood cell counts, and there were no changes in
`markersofliver toxicity (Supplementary Figure S2). However,clin-
`ical AAV2 administration has previously been associated immune
`recognition of AAV capsid peptides,” and therefore we evaluated
`cellular and humoral immunological recognition of AAV2.5.
`
`Cell-mediated immunity to AAV2.5
`Peripheral blood mononuclearcells (PBMCs) ofthe subjects were
`tested for recognition of potential AAV capsid and minidystrophin
`peptide epitopesby Elispotassay.” Antigens used to stimulate these
`responses were pools of overlapping peptides spanning the AAV
`
`
`
`Table 2 Theclinical data in patients with AAV2.5/minidystrophin musculardelivery
`Revertant
`Gene
`Time of Vector
`Placebo
`Mass
`Steroid
`Pre-Nab
`Vector
`
`control genome expression_fibersCapsid dose? biopsy
`
`
`(kg)
`use
`titer
`dose*
`Subject
`Age Deletion
`Saline
`D43
`Low
`0.75
`ND
`30-125
`1
`8
`45
`22.6
`Y
`<1:2
`6.0 x 10"
`6.6 x 10"
`Low
`6.6 x 10"!
`Low
`6.6 x 10"
`Intermediate
`3.3 x 10"
`
`2
`
`3
`
`4
`
`9
`
`9
`
`5
`
`50
`
`46-50
`
`49-54
`
`28.5
`
`35.5
`
`15.8
`
`Y
`
`N
`
`N
`
`1:800
`
`6.0 x 10!!
`
`Saline
`
`<1:2
`
`<1:2
`
`6.0 x 10"
`
`Saline
`
`3.0 x 10”
`
`Saline
`
`D90
`
`D43
`
`D43
`
`0.01
`
`0.61
`
`2.56
`
`ND
`
`weak
`
`0-9
`
`0-9
`
`ND
`
`0-11
`
`5
`
`ll
`
`3-17
`
`57.1
`
`y
`
`1:100
`
`3.0 x 10"
`
`Empty capsid High
`6.6 x 10”
`
`D90
`
`0.08
`
`ND
`
`ND
`
`3.0 x 10”
`Liz
`Y
`28.7
`46-52
`9
`6
`1-25
`weak
`1.42
`D43
`Empty capsid High
`6.6 x 10”
`Abbreviations: AAV, adeno-associated virus; ND, none detected; qPCR, quantitative PCR.
`@AAV2.5 minidystrophin vector genome dose (vector genomes/patient). Total capsid dose (minidystrophin + empty capsid in subject’s 5 and 6) capsid particles/
`patient. Vector genome copy numberisolated per nucleus as determined by qPCR(skeletal muscle cells are multinucleated).
`
`448
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`www.moleculartherapy.org vol. 20 no.2 feb. 2012
`
`Sarepta Exhibit 1062, page 6
`
`Sarepta Exhibit 1062, page 6
`
`

`

`
`
`
`
`IFN-ySFC/10°PBMC
`
`
`
`
`
`IFN-ySFC/10°PBMC
`
`
`
`
`
`IFN-ySFC/10°PBMC
`
`Subject 4 |
`
`Subject 5
`
`Subject 6
`
`Clinical Evaluation of Custom-designed AAV Capsid in DMD Patients
`
`(227 AAVcapsid peptide pool 1
`WB AAVcapsid peptide pool 2
`
`Subject 2 |
`
`8 15
`
`
`Base
`
`30
`
`43
`
`53
`
`60
`
`90
`
`120 180 365 Base
`
`8
`
`15
`
`30
`
`43
`
`53
`
`60
`
`90
`
`120 180 365
`
`Figure 4 Temporal T cell response to AAV2.5 capsid. Two capsid peptide pools were comprised of peptides spanning the AAV2.5 capsid sequence.
`Eli