Limb-Girdle Muscular Dystrophy Type 2DGene Therapy Restores �-Sarcoglycan and

Associated ProteinsJerry R. Mendell, MD,1,2,3,4 Louise R. Rodino-Klapac, PhD,1,2,4 Xiomara Rosales-Quintero, MD,1,2,4

Janaiah Kota, PhD,1,2,4 Brian D. Coley, MD,2,5 Gloria Galloway, MD,1,2,4 Josepha M. Craenen, MD,1,2

Sarah Lewis,4 Vinod Malik, PhD,4 Christopher Shilling, MS,1,2,4 Barry J. Byrne, MD, PhD,6,7

Thomas Conlon, PhD,6,7 Katherine J. Campbell,8 William G. Bremer,8 Laurence Viollet, PhD,4

Christopher M. Walker, PhD,1,2,8 Zarife Sahenk, MD, PhD,1,2,3,4 and K. Reed Clark, PhD1,2,4

Objective: �-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D[LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions relatedto this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression.Methods: A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevismuscle was conducted. Control sides received saline. A 3-day course of methylprednisolone accompanied gene transfer withoutfurther immune suppression.Results: No adverse events were encountered. SGCA gene expression increased 4–5-fold over control sides when examined at6 weeks (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber sizewas increased in the 3-month subject. Adeno-associated virus serotype 1 (AAV1)-neutralizing antibodies were seen as early as 2weeks. Neither CD4� nor CD8� cells were increased over contralateral sides. Scattered foci of inflammation could be found,but showed features of programmed cell death. Enzyme-linked immunospot (ELISpot) showed no interferon-� response to �-SGor AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 subjects successfully achieved study aims,precluding the need for additional enrollment.Interpretation: The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy.Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design ofvascular delivery gene therapy trials.

Ann Neurol 2009;66:290–297

The sarcoglycans, alpha, beta, gamma, and delta, forma subcomplex of the transmembrane dystrophin-glycoprotein complex.1,2 Sarcoglycan mutations are in-herited as autosomal recessive disorders, responsible forforms of limb-girdle muscular dystrophy (LGMD2C,delta-SG; LGMD2D, alpha-SG; LGMD2E, beta-SG;LGMD2F, gamma-SG).3–5 Overall, the clinical spec-trum associated with sarcoglycan gene mutations over-laps the scope of severity associated with dystrophinmutations responsible for Duchenne and Becker mus-cular dystrophies.6 There is no treatment forsarcoglycan-related LGMDs, with the exception of rare

individuals reported to benefit from immunomodula-tory agents.7,8

In the study reported here, LGMD2D, alpha-sarcoglycan deficiency, was the target disease based onpreclinical studies showing promise for gene thera-py,9,10 although 1 study raised issues of toxicity relatedto overexpression.11 It is the most common of the sar-coglycanopathies in North America and in other partsof the world.3,12 Novel findings in this phase I/II clin-ical gene therapy trial provide promising results for atherapeutic approach using adeno-associated virus sero-type 1 (AAV1) to transfer the alpha-sarcoglycan gene

From the 1Department of Pediatrics, the Ohio State University, Co-lumbus, OH; 2Nationwide Children’s Hospital, Columbus, OH;3Department of Neurology, the Ohio State University, Columbus,OH; 4Center for Gene Therapy, the Research Institute at Nation-wide Children’s Hospital, Columbus, OH; 5Department of Radiol-ogy, the Ohio State University, Columbus, OH; 6Department ofPediatrics, University of Florida College of Medicine, Gainesville,FL; 7Powell Gene Therapy Center, Gainesville, FL; and 8Center forVaccines and Immunity, the Research Institute at Nationwide Chil-dren’s Hospital, Columbus, OH.

Address correspondence to Dr Mendell, Research Institute at Nation-wide Children’s Hospital, 700 Children’s Dr. Room WA3011, Co-lumbus, OH 43235. E-mail: Jerry.Mendell@nationwidechildrens.org

Potential conflict of interest: Nothing to report.

Received Mar 6, 2009, and in revised form Mar 19. Accepted forpublication Mar 25, 2009. Published online in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.21732

This study has been registered at Clinicaltrials.gov NCT00494195.

290 © 2009 American Neurological Association

(SGCA). The study employed a double-blind random-ized controlled design to remove bias in outcomes anal-yses. Muscle after gene transfer demonstrated sustained�-SG gene expression accompanied by full restorationof the sarcoglycan complex. Although the study planincluded low- and high-dose cohorts (n � 3 each), thefindings in the low-dose group precluded the need toexpose patients to higher viral doses. The immune re-sponse did not impose a barrier to prolonged gene ex-pression in this study. It is also the first clinical reportemploying the muscle-specific creatine kinase (MCK)promoter13 to improve the safety profile of gene trans-fer targeting muscle. In addition, the study reinforcespreclinical predictions that the AAV1 serotype providesrelative persistence of genomes in dystrophic muscle.14

Materials and MethodsStudy DesignSubject eligibility included established SGCA mutations ofboth alleles, ability to cooperate for testing, willingness topractice contraception during the study (if appropriate), neg-ative pregnancy test (for females), and no evidence of cardio-myopathy, diabetes, or organ system abnormalities of bonemarrow, liver, or kidney. Human immunodeficiency virusinfection, hepatitis A, B, or C, or known autoimmune dis-eases were exclusion criteria. Subjects could not take immu-nosuppressive drugs or corticosteroids during the trial, andwere required to be off treatment for 3 months prior to en-rollment.

Institutional review board–approved consent and assent(for ages 9 to 17 years) forms were obtained by the principalinvestigator (J.R.M.) and signed by parents and subjects.Pre–gene transfer immune studies included serum neutraliz-ing antibodies to AAV1, interferon-gamma (IFN-�) enzymelinked immunospot (ELISpot) assay for both AAV capsidproteins and the �-SG protein.

This was a double-blind, randomized controlled trial ofrAAV1.tMCK.hSGCA vector injected into the extensor digi-torum brevis (EDB) muscle. This is a small muscle in thefoot that provides a unique site for gene expression, becauseit is relatively spared from the dystrophic process even in theface of advanced limb muscle degeneration. The study wasapproved by the Recombinant DNA Advisory Committee (#0610-815; October 31, 2006) and the Food and Drug Ad-ministration (FDA) (IND # is BB-IND 13434). Approxi-mately 4 hours prior to gene transfer, subjects received in-travenous methylprednisolone, 2.0mg/kg, used for its anti-inflammatory properties to reduce potential inflammationaroused by the needle manipulation at the time of genetransfer. The procedures were carried out in the pediatricintensive care unit at Nationwide Children’s Hospital underconscious sedation. The investigators received labeled sy-ringes (left and right) from the pharmacy, monitored fortemperature stability, containing either viral vector orphosphate-buffered saline (PBS). Injection sides were basedon computer-generated random numbers. Unblinding enve-lopes were held in the pharmacy. The gene delivery wasguided by ultrasound and electromyographic recordings(37mm Teca Myoject injection recording needle) to ensure

that muscle was the destination of the delivered product.Following injection, the empty syringes were resealed andstored at �20°C. Repeat doses of methylprednisolone weregiven at 24 and 48 hours postinjection. Patients received cor-ticosteroids only during this peri–gene transfer period, andreceived no other immunosuppressive drugs during the re-mainder of the trial.

EDB muscles were removed bilaterally from Patients 1and 3 at postinjection Days 42 and 51, respectively, andfrom Patient 2 at 12 weeks (92 days). The muscle was im-mediately cut into blocks of approximately 1.5 � 1cm, andfrozen in isopentane cooled in liquid nitrogen.

Vector ProductionrAAV1.tMCK.haSG was produced at the Harvard GeneTherapy Initiative according to current good manufacturingpractices. Vector production followed previously publishedmethods using plasmid DNA tritransfection of HEK293cells, followed by iodixanol and anion exchange columnchromatography purification.14 The vector was formulated insterile PBS, and passed all quality control acceptance criteriaestablished by the FDA for strength, identity, and purity.

Safety and EfficacyPost–gene transfer subjects were evaluated on the day of theEDB biopsies and Days 1, 2, 7, 14, 30, 60, 120, and 180.Photographs of the injection sites were taken immediately, at8 to 12 hours after gene transfer, and at each follow up visit.Efficacy was evaluated by blinded analyses of muscle tissueassessing �-SG gene expression by immune stains of musclesections and Western blot analysis with quantitation assessedby densitometry comparisons between sides. The number of�-SG–positive fibers was expressed as the percent of the totalnumber of fibers. “Total fibers” included only those fromblocks of muscle showing �-SG gene expression. This in-cluded 4 of 6 blocks in each case, with limitations imposedby spread of vector related to connective tissue barriers in themuscle and by the directional planes of injection illustratedin Figure 1. The results of the gene expression findings werepresented to the pharmacy and to our oversight Data SafetyMonitoring Board at the National Institutes of Health in awritten report before the blind was broken. Major histocom-patibility complex (MHC)-I and MHCII antigens were as-sessed on muscle sections. CD4� and CD8� mononuclearcells were reported as number/mm2 area. Muscle morpho-metrics included fiber size histograms.

Statistical analyses were based on differences between thesides in the total number of cells per square millimeter ofarea expressing CD4� and CD8� mononuclear cells,MHCI and MHCII antigens, and muscle fiber size using apaired t test (p � 0.05).

ResultsPatients and Dosing RegimenSix potential LGMD2D subjects met inclusion criteria.The risks, potential lack of benefits, and procedureswere explained prior to requesting informed consent.The 6 subjects were equally divided into low and highdose cohorts. Gene delivery to the EDB with subse-

Mendell et al: Gene Therapy for �-SG Deficiency 291

quent analysis has been completed in the 3 low-dosesubjects with the following characteristics: Patient 1,age 13 years, nonambulatory, homozygous for SGCAR77C substitution; Patient 2, age 12 years, nonambu-latory, homozygous for SGCA I124T substitution; Pa-tient 3, age 14 years, nonambulatory, compound het-erozygote with substitutions I124T/E137K. These 3subjects received 3.25 � 1011 vector genomes deliveredin 1.5ml of fluid in a 3ml syringe. The needle of thesyringe was inserted 0.5cm below the fascia (guided byultrasound) and pushed in the full length of the muscle(proximal to distal), parallel to the longitudinal orien-tation of the muscle fibers of the EDB. Additional con-firmation of muscle placement was obtained by elec-tromyographic recording in the muscle. The EDB liesin a groove on the proximal outer part of the foot. Thelateral head is prominently displayed with the proximalbase and apex pointing toward the small toe (Fig 1).The gene was delivered in equal amounts of fluid vol-ume (0.75ml) as the needle was withdrawn. The needlewas then reinserted 1

3the distance from base to apex

with delivery of the remaining fluid to muscle uponneedle withdrawal.

Safety of VectorPatient monitoring included vital signs hourly for 4hours and every 4 hours for the first 24 hours. The pa-tients were discharged and stayed locally until re-examination at 48 hours, after which time they weresent home. They returned for checkups according to theschedule provided (above). There were no adverse eventsof any kind. The site of injection never appeared swollenor erythematosus, and no rash was encountered. Therewas no difference observed between the side receivingvector versus saline. There was no pain in the postpro-cedure period. No organomegaly (liver, spleen) orlymphadenopathy (groin or axilla) was encountered.

Gene ExpressionAll analyses were done prior to breaking the blind. Rel-atively consistent gene expression findings were seenbetween subjects. Figure 2 shows findings by immunestains (A) and Western blots (B). Robust staining wasseen on only 1 side in each case that was easily distin-guishable from low level or background staining ob-served on the contralateral side. Photographs weretaken with adjustment of exposure times so that onlythe brightest fibers could be visualized, and counted.Quantitation revealed concordance between immune-stained sections and Western blots. On the side of ro-bust staining, 57% of fibers were positive in Subject 1,69% in Subject 2, and 62% in Subject 3. Sectionsfrom these same blocks taken for Western blots showeda 4- to 5-fold increase in all 3 cases. A further signof muscle repair was illustrated by restoration ofthe full sarcoglycan complex including �-, �-, and-sarcoglycan on the side of intense staining, easily dif-ferentiated from the contralateral side (Fig 2C). Subject2 demonstrated gene expression for 3 months (biopsywas done 92 days after gene transfer). There was nodiminution of staining comparing Subject 2 (at 3months) with Subjects 1 and 3 (6-week studies). Onthe side of increased �-SG expression, quantitativepolymerase chain reaction (PCR) demonstrated viralDNA gene transfer. A vector-specific primer probe setamplified a portion of the unique 5 untranslatedleader sequence of the �-SG cassette. SGCA transgenecopies increased by an average of 48-fold over baseline(representing 3.6 copies per nucleus) comparing theside of increased gene expression to the contralateralside. The RNase P gene was used as an internal controlto normalize for genomic input and confirm the ab-sence of PCR inhibitors in the sample DNA.

Muscle fiber size was determined for �-SG–positivefibers (n � 300 per side). After persistent gene expres-

Fig 1. (A) Extensor digitorum brevis (EDB) muscle shown with dotted lines indicating plane of injection from apex to base (longaxis) and across muscle from medial to lateral. (B) The ultrasound picture shows the injection needle inserted through the long axisof the muscle (arrow). The arrowheads (two above and two below) define the margins of the muscle.

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sion for 3 months, Subject 2 showed an increase inmean fiber diameter from 32.4 � 12.9�m to 45.4 �10.2�m, p � 0.001, comparing the side from whichvector was recovered with the opposite side. The genetransfer experiments studied at 6 weeks failed to showan increase in fiber size (Subject 1, control 57.6 �17.1�m vs treated 54.5 � 21.1�m, p � 0.18; Subject3, control 41.1 � 12.9�m vs treated 38.2 � 11.9�m,p � 0.07).

Immunologic Response to Vector-Mediated GeneExpression: Direct Muscle AssessmentMHC class I molecules were expressed on sarcolemmaof most muscle fibers on the side of upregulated geneexpression (Fig 3C). In contrast, MHC class II expres-sion was not observed. Mononuclear cell infiltrates

were evaluated on both sides in each of the 3 cases.The total numbers of CD8� and CD4� mononuclearcells per square millimeter of muscle was not differentbetween sides in any of the cases, although numberswere slightly higher on the side demonstrating in-creased gene expression (Fig 3A, B). In Subject 2, thebiopsy done at 12 weeks showed a few scattered clus-ters of CD4� and CD8� mononuclear cells, in theendomysial connective tissue and perivascularly. Thisraised the possibility of a focal reactive inflammatoryinfiltrate. Invasion of non-necrotic muscle fibers, acommon finding in MHC class I directed T-cell attackon muscle fibers in the inflammatory myopathies (in-clusion body myositis and polymyositis),15 was not ob-served. In spite of the focal infiltrate, �-SG gene ex-pression persisted without apparent loss as far out as

Fig 2. (A) Post–gene transfer tissue sections from extensor digitorum brevis muscles for Subjects 1, 2, and 3. The left panel shows�-SG gene expression on the side of vector injection (T) compared to muscle from the opposite side (C) (scale bar � 100�m). (B)Western blots from all 3 cases show increased �-SG gene expression on the side of gene transfer compared to the contralateral side(treated on left, control on right) showing residual gene expression from mutant protein. �-SG is normalized to actin (lowerband). (C) �-Sarcoglycan staining demonstrates restoration on the side of gene transfer. Other sarcoglycans (� and �) were alsorestored (not shown). Muscle from Subject 2 is shown (scale bar � 100�m). (D) Densitometry measurements show �-SG geneexpression increased 4- to 5-fold on the side of vector injection.

Mendell et al: Gene Therapy for �-SG Deficiency 293

the 3-month time point. This prompted us to furthercharacterize the inflammatory cells using a TUNELstain based on experimental gene transfer studies dem-onstrating programmed death of the antigen-specificeffector cells in AAV-transduced muscle.16 TheTUNEL stain showed that the majority of cells associ-ated with these foci of inflammatory cells appeared tobe undergoing apoptosis (Fig 3D).

Neutralizing Antibody to AAV1In all 3 subjects, serum neutralizing antibody titers toAAV1 were very low (1:100 or below) prior to genetransfer. Follow-up studies showed a slow rise in serumAAV1 titers that peaked at 6 to 12 weeks in Subjects 2and 3. This contrasted with the rapid rise and peakelevation by Week 1 in Subject 1, who also reachedserum titers 8-fold higher than the others (Table).

Table. Neutralizing Antibody Titers Against AAV1

TimePostinjection

Subject 1 Subject 2 Subject 3

Pretreatment 1:100 �1:50 1:100

1 wk 1:25,600 1:400 1:200

2 wk 1:25,600 1:3,200 1:1,600

6 wk 1:12,800 1:3,200 1:3,200

12 wk ND 1:3,200 1:3,200

26 wk 1:12,800 1:3,200 —

Endpoint titers determined using a cell-based reporter vectorinfection assay as previously described.25 AAV1 � adeno-associated virus serotype 1; ND � not done because samplenot available; — � to be collected.

Fig 3. (A) Compares the number of CD4� mononuclear cells/mm2 area between control and the side of gene transfer. (B) Com-pares the number of CD8� mononuclear cells/mm2 area between control and the side of gene transfer; (C) Major histocompatibil-ity complex class I (MHCI) staining of muscle sections shows lack of staining of muscle fibers on control (left) side, whereas the sideof gene transfer shows distinct staining of the sarcolemmal membrane in this subject (#3). Microvascular circulation is shown withMHCI staining on both sides (scale bar � 100�m). All 3 cases showed this same staining pattern on control and gene transfersides. (D) TUNEL-positive mononuclear cells (red) seen in a perivascular location in a muscle section from Subject 2. Other nucleiappear blue with 4,6-diamidino-2-phenylindole stain (scale bar � 100�m).

294 Annals of Neurology Vol 66 No 3 September 2009

Neutralizing antibody titers did not influence gene ex-pression.

ELISpot Assay for Detection of IFN-�Peripheral blood mononuclear cells (PBMCs) were se-rially assayed by ELISpot for antigen-specific produc-tion of IFN-� secretion, beginning before gene transferfor each subject (Fig 4). Patients 2 and 3 showed noIFN-� response to �-SG peptides or AAV1 capsid pep-tide pools. Subject 1 showed a very minimal IFN-�response specific to Pool 2 of the AAV1 capsid, exceed-ing our confidence limits for a negative response ( 50spot forming cells/1 million PBMCs) at Days 14 and43. This suggests a very low, transient T-cell–mediatedimmune response to capsid Pool 2 of AAV1 that wasunsustained.

PBMCs were further expanded by capsid peptidepool restimulation, and no additional AAV capsid re-sponses were detected. At no time was there evidenceof a T-cell response to the transgene product, �-SG.No anti–�-SG antibodies were detected using Westernstrip blots at baseline or at follow-up time points forany subject (data not shown).

DiscussionThe double-blind, randomized controlled trial designemployed in this study is uncommonly applied tophase I/II gene therapy trials. This provides an addedmeasure of confidence in our findings. On demonstra-tion of successful gene expression without adverseevents in Cohort 1, the blind was broken after discus-sions with the data safety monitoring committee. Byconsensus, the findings in the first 3 subjects made itunnecessary to expose 3 additional subjects to a higherdose.

In the patients under study, direct muscle injectionsof AAV1 delivering the SGCA gene under control ofthe truncated muscle-specific MCK promoter resultedin a 4-5–fold increase in �-SG expression for up to 3months. Although safety and gene expression were theprimary outcome variables, �-SG replacement resultedin restoration of the full sarcoglycan complex in allsubjects and an increase in muscle fiber size exclusiveto the subject showing gene expression for 3 monthsThe findings must be interpreted cautiously, but implyfunctional improvement based upon preclinical studieswhere increasing muscle fiber size, especially coupledwith gene replacement, shows protection againstcontraction-induced injury10,17 and increased gripstrength.18 Conventional functional measures of im-provement cannot be assessed in single-muscle humangene transfer trials and await gene delivery through thevasculature, allowing gene expression in multiple mus-cle groups and hopefully producing clinically meaning-ful results.

This is only the second completed trial using

AAV1,19 a serotype with potential advantages for ther-apeutic strategies employing direct muscle injection.20

A further novel aspect of this study is the use of thetruncated muscle-specific MCK promoter. In prepara-tion for this clinical trial, we systematically comparedSGCA gene expression using different promoters. Theconstitutive cytomegalovirus (CMV) promoter showeda trend toward reduced gene expression over time.10 Inanother study using CMV to express SGCA in the

Fig 4. Interferon-� (IFN-�) enzyme-linked immunospot assaysfor all 3 subjects. �-Sarcoglycan (�-SG) and control enhancedgreen fluorescent protein (eGFP) peptide stimulation showedno increase in spot forming colonies (SFC) per million periph-eral blood mononuclear cells (PBMCs) in any subject. In Sub-ject 1 there was a very minimal IFN-� response specific toPool 2 of the adeno-associated virus serotype 1 (AAV1) capsidexceeding our confidence limits for a negative response (�50spot forming cells/1 million PBMCs) at Days 14 and 43.Enzyme-linked immunospot assays were negative for Subjects 2and 3. dpi � days postinjection; red � �-SG; black �eGFP; blue � AAV1 capsid Pool 1; green � AAV1 capsidPool 2; purple � AAV1 capsid Pool 3).

Mendell et al: Gene Therapy for �-SG Deficiency 295

mouse, gene expression was lost over 4 to 6 weeks.11

For muscle gene therapy, the CMV promoter mayhave disadvantages because potential off-target gene-expression in antigen presenting cells could result ininadvertent augmentation of an immune response.

In light of the robust �-SG gene expression, thefindings observed in the gamut of immune studiesare particularly pertinent. Not surprisingly, AAV1-neutralizing antibodies in Subjects 2 and 3 appeared topeak at 2 weeks after gene transfer, followed by a pla-teau. Subject 1, found to have significant pre–genetransfer neutralizing antibodies to AAV2 (not observedin Subjects 2 or 3), generated a more rapid rise inAAV1 titers, reaching levels at least 8-fold higher com-pared to other subjects. This we presume to be an an-amnestic response related to cross-reactivity with theAAV2 capsid.

The IFN-� ELISpot assay showed low response lev-els considering the prediction based on other humangene therapy trials, where spot forming units/106 pe-ripheral blood mononuclear cells were far greater than100, indicative of a cytotoxic T-cell response.21 Directevidence of a transient AAV1 capsid-induced responsewas seen in Subject 1 that just exceeded our thresholdfor significance. However, attempts to enhance this byrestimulation with AAV1 peptide pools in Subject 1, aswell as parallel efforts in other subjects, did not showdetectable capsid-specific T-cell immunity. The up-regulation of MHCI in the muscle fibers on the side ofgene transfer suggests antigen presentation for a re-cently acquired epitope. Based on previous studies ofAAV2 Factor IX delivery through the portal vein,21

where a T-cell response was directed against the virus,it seems likely that the MHC response was a reflectionof the same process. In fact, this seems to correlatewith our findings of scattered foci of inflammatorycells seen on the side of gene transfer, especially inSubject 2. Of particular interest is that this inflamma-tion in no way precluded gene expression. Second, theinfiltrating cells showed findings consistent with pro-grammed cell death, suggesting that T cells were re-cruited to the scene but failed to invade the antigen-presenting target. A similar scenario for silencingantigen-specific T cells has been described in vector-transduced mouse muscle, with the majority ofantigen-specific CD8� T cells showing loss of functionand programmed cell death resulting in stable gene ex-pression.16 This could explain why T-cell infiltrationfailed to clear transduced muscle fibers in this clinicalstudy. In general, these findings contrast with loss ofgene expression following AAV2 Factor IX deliverythrough the portal vein.21 Further studies will be re-quired to sort out differences between observations, in-cluding the target tissue, route of delivery, and doseand serotype of AAV. The need for more aggressiveimmunosuppression for �-SG gene transfer can only

be assessed with clinical intramuscular or vascular de-livery gene therapy trials of longer duration.

In summary, this is the first gene therapy trial inmuscular dystrophy demonstrating promising findings,setting the stage for moving forward with treatment ofthis catastrophic group of diseases. Prior studies sup-port this viewpoint, showing both safety and persis-tence of AAV transcripts in nondystrophic skeletalmuscle.22 Nevertheless, clinically meaningful outcomeswill require vascular delivery to multiple musclegroups. Relevance beyond muscular dystrophy is alsoillustrated, because skeletal muscle offers a substrate forvector delivery of transgene products such as myostatininhibitors that could benefit a variety of neuromusculardisorders.18,23 The value of the EDB muscle as a safetesting site for initial gene therapy trials and a potentialdelivery site for secretory gene products was also dem-onstrated. A detailed description and method of injec-tion have been included in this discussion, because thelandmarks of the EDB are little known beyond thesubspecialty of electromyography and could be of valueto gene therapists. This target for gene therapy waspreviously advocated but never tested.24

This work was supported by the National Institute of Arthritis,Musculoskeletal, and Skin Diseases, National Institutes of Health1U54NS055958 (JRM) and the Muscular Dystrophy Association,and performed under the FDA IND # is BB-IND 13434 (JRM).Jesse’s Journey (JRM) also provided support.

William M. Fountain IV was responsible for stabilityassays on the vector; Hugh A. Allen, MD, providededitorial assistance; Richard C. Mulligan, PhD andJeng-Shin Lee, MD, PhD produced the clinical gradeAAV vector at the Harvard Gene Therapy Initiative,Department of Genetics, Harvard Medical School;Xiao Xiao, PhD, Department of Pharmacology, Uni-versity of North Carolina, kindly provided the trun-cated MCK promoter; Kevin P. Campbell, PhD, Uni-versity of Iowa, originally cloned the 50kDa SGCAused for the toxicology study to obtain the investiga-tional new drug and used in the gene construct deliv-ered to subjects in the clinical trial.

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