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Pediatric Neurology 51 (2014) 607e618

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Pediatric Neurology

journal homepage: www.elsevier .com/locate/pnu

Topical Review

Gene Therapy for Muscular Dystrophy: Moving the Field Forward

Samiah Al-Zaidy MD, Louise Rodino-Klapac PhD, Jerry R. Mendell MD *

Department of Pediatrics, Center for Gene Therapy, The Research Institute of Nationwide Children’s Hospital, Columbus, Ohio

* Commuof PediatrNationwid

E-mail a

0887-8994/$http://dx.doi

abstract

Gene therapy for the muscular dystrophies has evolved as a promising treatment for this progressive group ofdisorders. Although corticosteroids and/or supportive treatments remain the standard of care for Duchennemuscular dystrophy, loss of ambulation, respiratory failure, and compromised cardiac function is the inevitableoutcome. Recent developments in genetically mediated therapies have allowed for personalized treatments thatstrategically target individual muscular dystrophy subtypes based on disease pathomechanism and phenotype. Inthis review, we highlight the therapeutic progress with emphasis on evolving preclinical data and our ownexperience in completed clinical trials and others currently underway. We also discuss the lessons we have learnedalong the way and the strategies developed to overcome limitations and obstacles in this field.

Keywords: gene therapy, muscular dystrophy, exon skipping

Pediatr Neurol 2014; 51: 607-618

� 2014 Elsevier Inc. All rights reserved.

Introduction

The revolution in genetics, witnessed over the past fewdecades, has transformed the practice of medicine. Genetherapy exemplifies themove toward personalizedmedicine,where molecular-based therapies are tailored to the in-dividual’s genetic disorder. Approaches to gene therapyinclude direct gene replacement or gene repair or indirectlythrougha surrogate gene that enhances cellular performance.The promising findings from preclinical proof-of-conceptstudies in Duchenne muscular dystrophy (DMD) and thelimb-girdle muscular dystrophies (LGMDs) have moved thefield forward for the treatment of neuromuscular disorders.

In this review, we highlight the progress and lessonslearned with an emphasis on our own experience in clinicaltrials that are underway or have been completed.

Gene repair through mutation-specific therapies in DMD(nonviral vectors)

Exon skipping using antisense oligonucleotides

Mutations in the DMD gene disrupt the open readingframe and result in an incomplete translation of the

nications should be addressed to: Dr. Mendell; Departmentics; Center for Gene Therapy; The Research Institute ofe Children’s Hospital; Columbus, Ohio 43205.ddress: Jerry.Mendell@nationwidechildrens.org

- see front matter � 2014 Elsevier Inc. All rights reserved..org/10.1016/j.pediatrneurol.2014.08.002

dystrophin protein. Exon skipping is an approach to generepair that targets the pre-messenger RNA transcript,introducing alternative splice sites that result in skippingone or more targeted exons with resultant restoration ofthe dystrophin reading frame. Antisense oligonucleotides(AONs) are synthetically modified strands of nucleic acids,typically 20-30 nucleotides in length, composed of com-plementary sequences to dystrophin pre-messenger RNA.This treatment approach was inspired by evidence ofrestoration of the open reading frame of the dystrophingene expressed as “revertant fibers” by intrinsic alternativesplicing appearing in a high percentage of DMD boys.1 Theresult is small clusters of dystrophin-positive fibers, toofew to provide clinical efficacy. Synthetically designedAONs were designed to simulate naturally occurringalternative splicing, with the intention of achieving this in ahigher percentage of muscle fibers that could attain aclinically meaningful result. It is an approach that couldapply to approximately 83% of all DMD patients.2 The earlyemphasis for clinical trials has been to target the hot spotregion for deletions between exons 42 and 55 where mu-tations are generally amenable to exon skipping. Exon 51skipping is applicable to the largest group of all DMD pa-tients (13%) inclusive of deletions: 45-50, 47-50, 48-50, 49-50, 50, 52, or 52-63.2 Preclinical efficacy of AONs has beendemonstrated in the mdx, dystrophin and/or utrophinknockout (KO) mouse, and the dystrophin-deficient dogusing 20O-methyl-ribo-oligonucleoside-phosphorothioate

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and phosphorodiamidatemorpholino oligomers (PMOs).3-5

These two oligomers share a commonmechanism of actionbut differ in their biochemical structure, stability againstendonucleases, and toxicity profiles.

Two phase 1, proof-of-principle, clinical trials using a20O-methyl-ribo-oligonucleoside-phosphorothioate olig-omer (PRO051/GSK2402968; Prosensa Therapeutics) or aPMO (AVI-4658/Eteplirsen; Sarepta Therapeutics) targetingexon 51 were completed by a direct muscle injection, bothdemonstrating expression of dystrophin fibers.6,7 Safetyprofiles were comparable, with neither revealing adverseevents. These studies led to subsequent phase 2a open-labeltrials, in which higher doses of systemically delivered AONswere given to study participants. In the PRO051 trial, 12patients participated in a dose-escalation 5-week trial ofproduct given by subcutaneous injection.8 Dystrophinexpression was observed in approximately 60-100% ofmuscle fibers reaching 15.6% of normal. A 12-week exten-sion study improved the 6-minute walk distance by about35 m. Complications included injection site reactions andproteinuria without the loss of renal function. In the phase2, 12-week, open-label, dose-escalation PMO study (doserange, 0.5-20.0 mg/kg), dystrophin expression at thesarcolemmawas modestly increased in seven of 19 patientsto a mean of 8.9-16.4%.9 One patient reached 55% dystro-phin positive fibers after treatment with 20mg/kg. No drug-related adverse events were encountered.

The first, double-blind, randomized, controlled trial ofexon skipping using a PMO (eteplirsen; Sarepta Therapeu-tics) took place at Nationwide Children’s Hospital in Co-lumbus, Ohio.10 Enrollment included 12 DMD boys, ages 7 to13 years with confirmed of frame DMD gene deletions

FIGURE 1.Eteplirsen exon skipping study design. Twelve patients with Duchenne musculain study 201: cohort 1, received 30 mg/kg/wk; cohort 2, 50 mg/kg/wk; andtreatment, either 30 or 50 mg/kg/wk; thereafter referred to as “placebo delayeder the open-label extension study 202. Biceps biopsies were obtained on all paexpression. At week 12, biceps biopsies were obtained from patients in cohort 2biceps biopsies were obtained from patients in cohort 1 (30 mg/kg/wk) and tw

potentially correctable by skipping exon 51 that were ran-domized to one of three eteplirsen-treated cohorts: cohort 1,received 30 mg/kg/wk; cohort 2, 50 mg/kg/wk; and cohort3, placebo treated. At week 25, cohort 3 switched to open-label treatment, either 30 or 50 mg/kg/wk, thereafterreferred to as “placebo delayed” (Fig 1). All were on a stabledose of corticosteroids for 6 months minimum beforeenrollment. Outcome measures included dystrophin pro-duction, both percentage of muscle fibers and intensity atthe sarcolemma. Muscle biopsies were performed at base-line, at 12 weeks for the 50 mg/kg cohort and 24 weeks forthe 30 mg/kg cohort. Two blinded placebo-treated patientswere biopsied at 12 weeks and two at week 24. At the week25, the placebo-treated patients (n ¼ 4) received weeklyeteplirsen dosing, two received 30 mg/kg and two received50 mg/kg. All patients had a third biopsy at week 48. The 6-minute walk test (6MWT) was the primary functionaloutcome, performed pretreatment and posttreatmentthrough week 48.

The results of dystrophin production at all time pointsare illustrated in (Fig 2). At 12 weeks after treatment, nosignificant dystrophin was produced (50 mg/kg cohortand two placebo treated). Dystrophin production could beobserved after 24 weeks of eteplirsen except for the twoplacebo-treated patients (as expected). At week 48, allpatients, including the placebo-delayed cohort, producedsignificant dystrophin. These findings are a clear indica-tion that dystrophin production started sometime afterweek 12 and was more influenced by duration of treat-ment than dose. We also found restoration of the sarco-glycans and nNOS-binding site accompanying dystrophinproduction.

r dystrophy were randomized (R) to one of three eteplirsen-treated cohortscohort 3, placebo treated. At week 25, cohort 3, switched to open-labeld”. Patients were maintained on the same starting dose of eteplirsen un-tients at baseline and deltoid biopsies at week 48 for analysis of dystrophin(50 mg/kg/wk) and two placebo-treated patients in cohort 3. At week 24,o placebo-treated patients in cohort 3. Reprinted from Mendell, et al.10

FIGURE 2.Effects of eteplirsen on dystrophin expression. (A) Representative example of time dependent increases in dystrophin-positive fibers throughout the studycan be observed. Dystrophin expression begins between week 12 and week 24 of eteplirsen treatment and continues to increase after that time point. (B)Western blot from a representative patient demonstrates measurable dystrophin at week 48 compared with baseline. (C) reverse transcription polymerasechain reaction (RT-PCR) demonstrates the presence of skipped messenger RNA product (289 bp) after eteplirsen treatment in the muscle of a representativepatient with exons 49-50 deletion. Pt, patient; Tx, treatment. Reprinted with permission from Mendell, et al.10

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The 6MWT results for the four patients in the placebocohort were consistent with natural history studies11,12

resulting in decline of almost 70 m from baseline by week48. In the treated cohorts from study initiation, analysis wascomplicated by twin boys in the 30 mg/kg cohort whorevealed rapid disease progression. By week 24, whenconsistent increases in dystrophin positive fibers wereobserved, one of the twins lost ambulation and the otherwas teetering on loss. In retrospect, their baseline profilepredicted imminent loss of ambulation (the tallest andamong the oldest of the 12 patients enrolled and had thelowest baseline 6MWT values).11,13 The severe deviationfrom normality because of their extreme values placedthem in an “outliers” category justifying exclusion from theearly treatment group. Thus, we compared the sixambulation-evaluable patients in the 30 mg/kg (n ¼ 2) and50 mg/kg (n ¼ 4) cohorts who received eteplirsen for48 weeks and demonstrated stable performance on the6MWT from baseline to week 48. This was significantlydifferent (67.3 m difference; P � 0.001) compared with theplacebo-delayed cohort (Fig 3).

The findings from this study reveal a clear relationshipbetween systemic treatment with eteplirsen, an exon-skipping drug, and demonstration of dystrophin productionin DMD muscle, followed by a statistically significant differ-ence in walking ability in treated versus placebo-delayedpatients. Eteplirsen’s clinical benefit mirrored the timerequired to produce consistent increases in dystrophin. Thisobserved delay between initiation of eteplirsen and detect-able dystrophin production and stabilization in clinicalfunction likely results from an accumulation of novel dys-trophin over time. It also has to be underscored that thesafety profile of eteplirsen could not be any better as not a

single significant adverse event has been attributable to thisagent. The study has now extended for more than 1.5 years,and stability and safety continue without deviation.

This very promising method of treatment is nowspreading to other exons. The effect of skipping differentexons would be expected to vary given the binding sites andfolding of smaller dystrophin proteins. Nevertheless, this isa promising mode of therapy that is moving forward.

Suppression of nonsense mutations

Aminoglycosides (gentamicin)

Premature termination codons (PTCs) because ofnonsense mutations account for approximately 13-15% ofall DMD mutations.14,15 As with exon skipping, stop codonreadthrough is also mutation specific. The end product ispotentially a full-length dystrophin protein. Biochemicaland molecular studies of readthrough suggest that amino-glycosides bind a specific site in ribosomal RNA and impaircodon-anticodon recognition at the aminoacyl-transferRNA acceptor site. This introduces a missense mutationpermitting translation by ignoring the premature stopcodon. An important question is whether this finding couldbe harnessed for translational benefit to patients. Proof-ofprinciple studies were first demonstrated using amino-glycosides to treat the cystic fibrosis mouse model restoringthe full-length cystic fibrosis transmembrane conductanceregulator protein.16,17 Studies were extended to the mdxmouse, where the aminoglycoside, gentamicin, successfullyreduced creatine kinase (CK) levels by 70% and increaseddystrophin expression to about 20%.18 Gentamicin-treatedmice revealed improved histology and protection against

FIGURE 3.Functional efficacy of eteplirsen. The purple line illustrates the change from baseline (BL) in distance walked on the 6-minute walk test (6MWT) over timefor the six evaluable patients who received eteplirsen from the start of study 201. The dark gray line represents change from baseline in distance walked onthe 6MWT for the four patients who received placebo for the first 24 weeks and eteplirsen for the last 24 weeks. It is estimated from dystrophin expressionstudies and multiple biopsies that significant dystrophin was produced after 12 weeks. This is clearly indicated in the placebo/delayed cohort starting oneteplirsen at week 25 and stabilizing in function at week 36. Eteplirsen-treated cohorts beginning at study initiation maintained a plateau without declinethrough week 48, diverging from placebo/delayed patients at week 12. Two of the treated patients starting at week 1 lost ambulation before they producedsignificant dystrophin and are not represented on this graph. Tx, treatment. Reprinted with permission from Mendell, et al.10

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contraction-induced injury. The encouraging preclinicalstudies served as a template for a similar approach in DMDpatients with confirmed nonsense mutations in the dys-trophin gene. The initial clinical trial reported in 2001involved two DMD and two Becker muscular dystrophy(BMD) patients who received 7.5 mg/kg intravenousgentamicin for 2 weeks19 with a drop in serum CK, butrestoration of dystrophin was not observed at this earlytime point. A subsequent trial conducted in a small cohort offour DMD patients reported more favorable results withgentamicin given over two cycles, each for 6 days with a 7-week interval between dosing.20 Dystrophin expressionwas observed in three of four patients’ muscle biopsies, butno clinical meaningful outcomes were obtained in any ofthe patients.

A third clinical trial conducted by our group at Nation-wide Children’s Hospital consisted of a two-phase proto-col.21 The first phase was intended to establish biopotencyin a short-term 14-day dosing study, simulating what hadbeen performed in mdx mice and ensuring adequatetranslation to the clinic before an extended 6-month trial.Ten DMD boys with proven stop codon mutations wererecruited for daily gentamicin administration using a stan-dard dosing regimen (7.5 mg/kg) for treatment of gram-negative infections. Serum CK served as the primaryoutcome measure and both a biologic effect and a 50%reduction of CK at day 14 were established. Havingdemonstrated biopotency in the short-term trial, a six-month study was designed using muscle strength andfunction as outcomes for clinical assessment. The dosingregimen for gentamicin was dictated based on the knownprolonged half-life of dystrophin, with estimates of as longas 6-8 weeks22,23 justifying weekly and twice-weeklydosing regimens. Enrollment was contingent on normalhearing and kidney function, and negative testing for the

known A1555G mitochondrial DNA mutation in the 12Sribosomal RNA gene predisposing to aminoglycoside-induced hearing loss.24 Renal function was monitored us-ing cystatin C to avoid false-negative findings related todecreased serum creatinine and creatinine clearance levelsin this population.25

Sixteen patients with documented stop codon mutationsreceived weekly (n ¼ 12) or twice-weekly (n ¼ 4) genta-micin. No patient demonstrated a decline in renal or hear-ing function except one patient given a miscalculated dose(125% of recommended) for the first four administrationsresulting in a transient high-frequency hearing loss thatreturned to normal within 3 months. In nine patients,pretreatment dystrophin levels ranged from 0.8% to 4.5%;six of which indicated increased levels after 6 months ofgentamicin treatment. In three patients, dystrophin proteinincreased to a potentially therapeutic range with indepen-dent concordance by immunofluorescence (Bioquant imageanalysis) and Western blot analyses. The most significantreadthrough was observed in three patients increasingdystrophin levels to 13.0%, 15.4%, and 15.4% of wild-typelevels (Fig 4). No conclusion could be reached regardingpreferential stop codon predicting readthrough or relationto the fourth base surrounding the stop codon as suggestedby others.26

In addition to dystrophin expression in muscle tissue,serum CK was mildly reduced after 6 months of gentamicintreatment (9851 � 2109 U/L to 5316 � 850 U/L, P ¼ 0.04).However, functional outcomes failed to improve includingtime towalk 30 feet, or climb four standard steps, and standfrom supine position on the floor.

Overall, the clinical trial validates preclinical observa-tions in mdx mice revealing that mutation suppression orreadthrough can be translated to patients. However, onlythree patients in this clinical trial reached dystrophin levels

FIGURE 4.Dystrophin expression after gentamicin treatment. (A and B) Pregentamicin and postgentamicin treatment muscle biopsies demonstrating dystrophinexpression by immunofluorescence (IF) using DYS2 Novacastra; adjacent Western blot (WB) reveals full-length dystrophin at 427 kDa. Gentamicin-induceddystrophin expression increased to 15.44%. (C and D) Pretreatment and posttreatment muscle biopsy; IF sections stained with DYS2 antibody; adjacent WBreveals full-length dystrophin at 427 kDa. Gentamicin-induced dystrophin increased to 13.0%. (E and F) Pretreatment and posttreatment muscle biopsysections from a patient without response to gentamicin indicating negligible increase after treatment by IF and WB. Scale bar ¼ 100 mm. Reprinted withpermission from Malik, et al.21

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that could potentially slow the progression of the disease.Results could theoretically be improved by increaseddosing, but the risk-benefit considerations of aminoglyco-side therapy must be weighed in favor of agents that havepotentially fewer side effects and can be administeredorally, as discussed in the next section.

PTC124 (Ataluren)

Ataluren is a small molecule developed by PTC Thera-peutic in an effort to advance an orally bioavailable productthat selectively bypasses disease-causing nonsense muta-tions without an effect on stop codons and avoids the po-tential renal and ototoxicity of aminoglycosides. It wasoriginally developed by means of an optimized high-throughput screening campaign. Dose-dependent read-through of all three nonsense codonswas observedwith thehighest readthrough at UGA, followed by UAG and thenUAA. Compared with gentamicin, PTC124 demonstratedhigher potency of nonsense suppression at lower concen-trations.27 Treatment of mdx mice restored dystrophinproduction in all skeletal muscles examined, including thediaphragm, and cardiac muscle within 2-8 weeks of expo-sure. A significant reduction in serum CK levels wasobserved after 2 weeks of treatment. The dystrophin levelswere evident to be 20-25% of control mouse muscles and

partially restored force generation and resistance againsteccentric exercise.

In two phase 1, dose-escalating, safety, and tolerabilitystudies, PTC124 was well tolerated with the exception ofmild headaches, dizziness, and gastrointestinal discomfortat higher doses.28 A phase 2b, double-blind, placebo-controlled, dose-ranging, efficacy, and safety study failed todemonstrate improvement in the 6MWT.29 Further detailedanalysis of the phase 2b data, however, suggested the pos-sibility of a bell-shaped dose response with clinical benefitto DMD boys receiving low dose and in high-dose patientsin whom serum levels of PTC124 were low. In view of thesetentative findings, PTC124 has moved ahead with a phase 3,multicenter, randomized, double-blind, placebo-controlledstudy to determine efficacy and safety of Ataluren in DMDboys aged 7-16 years with known stop codons. The study iscurrently underway, and results are not yet available(ClinicalTrials.gov identifier: NCT01826487).

Gene replacement through viral vector-mediated therapies inmuscular dystrophy

Duchenne muscular dystrophy

DMD, the most common childhood muscular dystrophy,is an X-linked recessive disorder because of mutations in

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the dystrophin gene. Currently, the only treatment withproven efficacy is corticosteroids. Although a detailed re-view of corticosteroid trials is beyond the scope of this re-view, it is pertinent to emphasize the unequivocal evidenceof functional benefit from this class of drugs. Prolongedambulation was well documented in a randomized, double-blind, controlled trial of daily prednisone versus placebo.30

Treatment with corticosteroids has become standard of careand a requirement for enrollment in most clinical trials.Several regimens for initiating steroid treatment are usedworldwide with some variability in the adverse effect pro-file of each regimen.31

Dystrophin, the largest gene in the human genome, iscomposed of 79 exons encoding an approximately 427 kDsubsarcolemmal protein that is widely expressed in bothskeletal and smooth muscles. Spontaneous mutations leadto a high incidence of disease in newborn males estimatedat approximately1:5000.32,33 Using a gene replacementapproach potentially circumvents restricted applicability ofmutation-specific gene repair therapies. However, the largesize of the dystrophin gene impedes packaging into viralvectors considered to be the most suitable for gene deliveryto skeletal muscle and cardiac muscle. In an effort to over-come these hurdles, two promising strategies emerged inthe field: functional miniaturization of dystrophin that canfit into recombinant adeno-associated virus (rAAV) andvascular delivery that allows for a wider distribution oftherapeutic gene.

The concept of miniature forms of the dystrophin genethat could translate into a functional protein came from theclinical observation of a BMD patient who remainedambulatory until the seventh decade, in spite of a deletionin 46% of his DMD gene.34 Starting with this as a template,mini- and micro-dystrophin constructs were engineered toencode integral domains of the dystrophin protein suffi-cient to produce a functional protein. Preclinical studies inthe mdx mouse using a mini-dystrophin gene (D3990 kb)under control of a cytomegalovirus (CMV) set the stage fortranslation.35,36 Robust and sustained gene expression wasobserved for 6 months, with protection of muscle fibersfrom uptake of Evans blue dye and an increase isometricforce and resistance to contraction-induced injury.

In a first clinical gene therapy trial of its kind in DMD, sixboys were enrolled in a double-blind, randomized,controlled, dose-escalation study evaluating safety and ef-ficacy of dystrophin gene transfer.37 All six patients hadconfirmed frameshifting deletions of the dystrophin gene;four were on corticosteroid treatment that continuedthroughout the study. The vector used in this trial was amodified AAV serotype 2 capsid with an insertion of fiveamino acids from AAV1 encapsulating a functional mini-dystrophin transgene, representing approximately 40% ofthe 11-kb coding sequence of the human dystrophin gene(rAAV2.5.CMV.D3990). This was a dose-escalation trial; thebiceps muscle of one side received the therapeutic gene,whereas the contralateral side received either saline (cohort1, n ¼ 3) or empty capsids (cohort 2, n ¼ 3).

Gene transfer efficiency was evaluated by examiningbiopsy specimens of vector-injected versus untreated bi-ceps muscles on day 42 and day 90. Expression of the mini-dystrophinwas only detectable in a fewmyofibers from twoof the four treated muscle samples evaluated 42 days after

injection of two patients. Neither the treated samples takenat day 90 after injection nor the untreated side revealed anyevidence of transgene expression. An important aspect ofgene therapy trials is safety, and in this trial, no adverseevents were encountered.

In spite of failure to achieve successful transgeneexpression, this trial proved to be informative with regardsto the impact of cellular immune responses on theoutcome of gene replacement therapy. Peripheral bloodmononuclear cells (PBMCs) were tested in an interferon(IFN)-g enzyme-linked immunosorbent spot (ELISpot)assay for reactivity to mini-dystrophin. Three syntheticpeptide pools spanning the entire mini-dystrophinsequence (designated MDP1, MDP2, and MDP3) wereused to stimulate PBMCs in the ELISpot assay. One patientshowed a robust T-cell response against MDP2 beginningon the fifteenth day after gene transfer (Fig 5). Theresponse peaked on day 30, slowly declining during thenext 3 months, and was intermittently positive after day120. In cohort 2, two patients had positive ELISpots toMDP3. Additional studies revealed that at least twodifferent pathways provoked an immune response tomini-dystrophin. In one patient, the delayed, transient, T-cell response detected discrete dystrophin epitopes, all inthe region of the patient’s deletion. Because exons 3through 17 were deleted from the DMD gene in this pa-tient, it was clear that CD4þ and CD8þ T cells were primedby mini-dystrophin that expressed exons 3 through 12 inthe region of the patient’s deletion (Fig 5). In one patient, adifferent and unexpected mechanism of immunity waselicited. An MDP2-specific, IFN-g, T-cell response wastriggered by amino acids 2809-2829 of exon 57, down-stream from the patient’s exon-50 deletion. The patient’smutation predicted a frameshift, thereby impeding dys-trophin expression. However, in the muscle biopsy fromone patient, revertant fibers were visualized with the useof exon-specific antibodies against dystrophin (Fig 5).Thus, T cells were primed from exon 57, and we were ableto demonstrate a robust IFN-g response in PBMCs not onlyafter gene transfer but also before treatment as well. Asimilar situation was observed in another patient who hada deletion at exons 49 through 54, in whom we foundrevertant fibers with novel epitopes encoding exon 59,downstream of the mutation.

The findings from this study are important as we moveforward with gene therapy for DMD. First, we learned thatwe could not express mini-dystrophin in a deleted region ofthe patient’s DMD gene. In retrospect, this seems intuitive,but it was underappreciated as inclusion and/or exclusioncriteria for this study. The second important lesson learnedis that revertant fibers, expressing dystrophin by restoringthe reading frame in the DMD gene through alternativesplicing or a second-site mutation, can express an immu-nogenic novel epitope never before encountered by thepatient, most likely related to misfolding of the newlyexpressed dystrophin. This finding was very much unex-pected given that most clinicians and scientists predictedthat revertant fibers would induce immune tolerance.Validation of this principle was demonstrated in a follow-up study to determine the prevalence of such immunitywithin a DMD cohort from our Muscular Dystrophy Asso-ciation clinic.38 Dystrophin-specific T-cell immunity was

FIGURE 5.Dystrophin-specific T-cell response. Peripheral blood mononuclear cells (PBMCs) were tested in an interferon (IFN)-g enzyme-linked immunosorbent spot(ELISpot) assay for reactivity to mini-dystrophin in two patients in the clinical Duchenne muscular dystrophy gene therapy trial. Three synthetic peptidepools spanning the entire mini-dystrophin sequence (designated MDP1, MDP2, and MDP3) were used to stimulate PBMCs interferon-g T cells in the ELISpotassay. (A) Patient with exon 50 deletion revealed a robust T-cell response against MDP2 beginning on the 15th day after gene transfer. The response peakedon day 30 and slowly declined during the next 3 months. (B) An unexpected mechanism of immunity was evident for this patient with exon 50 frame-shifting deletion. The immune response was triggered from amino acids 2809 to 2829 of exon 57, downstream from the patient’s deletion. Exon-specific antibodies against dystrophin revealed that exon 57 was expressed in the revertant cluster (antibody stains revealed expression of exons 55-56and 59 implying that exon 57 was also expressed). (C) In a different patient with exons 49 through 54 deleted, T cells were found directed against exon 59,downstream of the frame-shifting mutation. These patients illustrate that misfolded protein expressed on revertant fibers can trigger a T-cell immuneresponse. Reprinted with permission from Mendell, et al.37

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evaluated in patients with DMD who were either receivingcorticosteroids in the form of prednisone (n ¼ 24) ordeflazacort (n ¼ 29) or who were not receiving steroids(n ¼ 17), as well as from normal age-matched control pa-tients (n ¼ 21). Anti-dystrophin T-cell responses weredetected in none of the normal control patients, and re-sponses were present in nine of 17 (52.9%) untreated pa-tients with DMD. In contrast, theywere evident in only 11 of53 (20.8%) treated patients with DMD. These findingsconfirm the presence of preexisting circulating T-cell im-munity to dystrophin in a sizable proportion of patientswith DMD and emphasize the need to screen patients fordystrophin immunity before clinical gene therapy trials.

Limb-girdle muscular dystrophy type 2D

The alpha-sarcoglycan (a-SG) deficient subtype of LGMD(limb-girdle muscular dystrophy type 2D [LGMD2D]) is themost common of the four sarcoglycanopathies.39 This dis-ease shares many phenotypic similarities to DMD with theexception of its autosomal recessive inheritance pattern.Cognitive function is not affected, and the heart is most oftenunaffected. Clinically, the spectrum of disease is similar todystrophin deficiency including a severe DMD-like pheno-type and a milder form simulating BMD with preservedambulation into adulthood. Currently, there is no effectivetreatment for LGMD2D. In preparation for a clinical trial,

proof of principle studies were conducted at our center in thea-SG KO mouse using a muscle-specific CK promoter andrAAV1 serotype for intramuscular (IM) delivery.40 This choiceof vector construct allowed for robust and long-term geneexpression in the tibialis anterior muscle without signs ofinflammation or toxicity paving the path for clinical trial.

The LGMD2D clinical trial of a-SG gene transfer carriedthe responsibility to address the lessons learned fromthe prior DMD gene transfer trial and the concerns ofsafety. In a randomized, double-blind, control study,rAAV1.tMCK.hSGCA was injected into the extensor dig-itorum brevis (EDB) of six patients with geneticallyconfirmed LGMD2D.41 The contralateral EDB served as acontrol receiving saline injections. The investigators per-forming the injections were blinded to the side of genetransfer. The EDB injections provide a source for valuabletissue assessment after gene transfer, and removal of themuscle causes no loss of function to the patient. Coincidentwith gene transfer, to suppress an immune response, pa-tients received a 3-day course of methylprednisone start-ing 4 hours before gene injections with repeat at 24 and48 hours after injection.

In this trial, the patients were initially divided into low-and high-dose cohorts to allow for dose escalation. How-ever, sufficient evidence of successful gene transfer at6 weeks and 12 weeks at low dose (3.25 � 1011 vg) pre-cluded the need for a higher dose regimen in the

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remaining three participants. This second cohort of pa-tients received the same dose of vector but had geneexpression evaluated at a 6-month postinjection timepoint.42 In all six participants, the question of safety wasanswered with no adverse events recorded throughout thestudy. Unequivocal muscle fiber transduction of a-SG wasconsistently observed in both cohorts with the exceptionof one patient whose biopsy revealed no differences beforeand after injection (Fig 6). Accounting for this was an earlyrise in neutralizing antibodies at day 2 after gene deliveryand early T-cell responses to AAV1 capsid measured byIFN-g ELISpot assay (Fig 7). These early immune responsesfavored an amnestic response related to pre-existing im-munity to AAV.

This clinical trial helps provide the foundation for mov-ing forward with gene delivery of the a-SG transgenedispelling any concerns regarding toxicity. The safety profileis further enhancedwith the demonstration of gene transferusing a muscle-specific (tMCK) promoter by avoiding con-cerns of off-target gene expression. The trial also revealedthe importance of testing all patients for pre-existing im-munity to AAV; both neutralizing antibody levels and T-cellimmunity.

FIGURE 6.a-Sarcoglycan (SG) expression after gene transfer in limb-girdle muscular dystrodigitorum brevismuscles were stainedwith antibody to a-SG. Two patients exprespatient revealed no difference in a-SG staining intensity before and after gene traWestern blots reveal increaseda-SGgene expressionon the sideof gene transfer compatients; residual gene expression from mutant protein prominently exhibited in

Genetic enhancement of alternate genes to improve cellularperformance

Follistatin gene therapy

Blocking the myostatin pathway reveals dramatic musclebuilding properties that are highly conserved across spe-cies, includingmice, sheep, cattle, canines, and humans [62-64]. Myostatin is a member of the transforming growthfactor-b superfamily and numerous studies have demon-strated that it is a negative regulator of skeletal musclegrowth. Follistatin is a powerful inhibitor of myostatin plushaving the advantage of controlling muscle mass throughpathways independent of the myostatin signaling cascade[65]. In studies of the myostatin KO mice crossed to micecarrying a follistatin transgene, there was a quadrupling ofmuscle mass compared with the doubling of muscle massthat is observed from lack of myostatin alone.

In preparation for taking this to clinical trial, we identi-fied an alternatively spliced isoform of follistatin (FS344)that can be packaged in AAV1. In the mdx mouse, IM in-jections of AAV1 carrying the FS344 gene increased musclemass (histologic measure), hind limb and forelimb grip

phy type 2D patients. (A) After gene transfer, tissue sections from extensorsed increased staining on treated side (T) comparedwith control side (C). Onensfer (findings verified by Bioquant Image Analysis) (scale bar ¼ 150 mm). (B)paredwith the contralateral side (treatedon left and control on right) for two

another individual. Reprinted with permission from Mendell, et al.42

FIGURE 7.MHC class I staining of sarcolemmal membrane of muscle sections on treated (T) and control (C) sides. (A) Gene transfer leads to antigen presentation at thesarcolemma. The absence of MHC I in a patient in the limb-girdle muscular dystrophy type 2D (LGMD2D) study was in direct contrast to the findings of all otherparticipants in the clinical trial, illustrated by prominent MHC I expression demonstrated in a patient on the treated side. Blood vessels will reveal nonspecificpositive staining forMHC I gene. (B) A patient in the LGMD2D gene transfer study also had an immune response demonstrated in the interferon (IFN)-g enzyme-linked immunosorbent spot (ELISpot) assay to adeno-associatedvirus (AAV)1 capsidpool2 (AAV1CP2). Thiswasobservedasearlyasday2 (blue) andagainonday7. Responseswerenegative forothercapsidpools (AAV1CP1andAAV1CP3). Thepositive IFN-gELISpot assayoccurring shortlyaftergene transferwas indicativeofan amnestic response suggesting pre-existing immunity to AAV1. PBMC, peripheral blood mononuclear cell. Reprinted with permission fromMendell, et al.42

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strength, and quantitative measures force generation andprotection against eccentric contractions [65]. The benefitspersisted for more than 2 years, and there were no adverseeffects. In nonhuman primates, direct muscle injections ofAAV1.CMV.FS344 into the quadriceps muscle increased sizeand strength for as long as 15 months [66]. Necropsiesrevealed no abnormalities in any organ, and clinicalchemistries and hormone levels related to gonadal function

including follicle stimulating hormone, luteinizing hor-mone, testosterone, and estradiol were normal.

We have now taken this to clinical trial in two patientgroups who could specifically benefit by increasing size andstrength of the quadriceps muscle. In BMD and sporadicinclusion body myositis (sIBM), the quadriceps muscle istargeted by the disease leading to frequent falls and loss ofambulation. In the gene therapy trial, the first three patients

FIGURE 8.AAV5.Dysf gene delivery. Dysferlin-deficient mice (Dysf�/�) mice were treated by intramuscular or vascular delivery via the femoral artery with AAV5.Dysf.Dysferlin expression was restored 1 month after treatment (right panel) compared with untreated mice (left panel). The overexpression of dysferlin in themuscle fibers is typical of this method of gene transfer and is nontoxic to the cell. AAV, adeno-associated virus; Dysf, dysferlin. Reprinted with permissionfrom Grose, et al.54

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(sIBM) were injected with AAV1.CMV.FS344 at low-dosevector (2 � 1011 vg/kg) in a single limb required by theFDA. No adverse events were encountered, and patientstransiently improved the distance walked on the 6MWT (inspite of unilateral injections). In the second group, threeBMD patients received an intermediate dose (6 � 1011 vg/kg). The distance walked on the 6MWT improved for 6months in two of three patients. Treatment has nowextended to the high-dose BMD group (n ¼ 3); bilateral IMinjections of 1.2� 1012 vg/kg have been injected. Results arestill under evaluation. The FDA has given approval for sixsIBM patients to receive this same high dose in both legs.The distance walked on the 6MWT is the primary outcomemeasure with secondary end points including muscle bi-opsy analysis and magnetic resonance imaging of quadri-ceps muscles.

Lessons learned and strategies to enhance gene delivery

LGMD2D vascular delivery

In most gene replacement trials for the muscular dys-trophies, proof-of-principle studies have used IM injections

to deliver the therapeutic gene. In an effort to achieve widerdistribution of gene expression, vascular delivery can beused to target single limbs or can be administered to pro-vide systemic distribution. Beyond the benefit of wide-spread distribution, vascular delivery holds severaladvantages. Most importantly, vascular delivery is lessimmunogenic compared with IM delivery.43,44 Immunoge-nicity results from increased vector genome copy numberper nucleus, approximately five to 10 times greaterfollowing direct IM muscle injections versus vascular de-livery. The goal of a more equal distribution of uniformprotein expression across the length of the muscle45 ac-companies vascular delivery.

Different strategies have been used in large animals todeliver the gene to isolated limbs through either the venousor arterial circulation.44,46-49 Our group has preferred to usean arterial approach that avoids the high-volume high-pressuremethod of gene delivery that is required for deliverythrough the venous circulation.50We have used fluoroscopy-guided catheterization to target individual muscle groups,such as the gastrocnemius and quadriceps, or to delivervector to the entire lower limb. To optimize arterial genedelivery and distribution, we have developed amethod using

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recirculation that improves vector transduction. This methoduses a closed circuit with a tourniquet placed at the level ofthe groin accompanied by catheterization of both thefemoral artery and vein. Venous blood is first removed fromthe circuit and stored in a reservoir followed by infusion of asaline-based solution to remove platelets and other cellsfrom the extremity that could bind to vector. The vector isthen infused and recirculated using a mechanical pump,thereby offering transgene to many muscle groups in theextremity throughmultiple passes. The promising preclinicalresults of this recirculation approach provided a rationale fortranslation to a clinical trial in LGMD2D patients. We havepermission from the FDA to move ahead with a limb recir-culation approach using AAV to deliver the a-SG transgene.This will be the first vascular gene therapy trial for musculardystrophy and be adapted to for treatment of DMD, LGMD2B,and other dystrophies.

LGMD2B homologous recombination

LGMD2B is an autosomal recessive form of musculardystrophy because of dysferlin (DYSF) deficiency. It repre-sents one of four allelic phenotypes that also includeMiyoshi myopathy with calf muscle weakness and atrophy,a distal anterior compartment myopathy with tibialismuscle atrophy, and a less common subtype with rigidspine syndrome.51-53 This spectrum of myopathies isbecause of mutations in the DYSF gene. The major challengefor gene therapy in dysferlinopathies is that the 6.5 kb DYSFcomplementary DNA exceeds the packaging capacity ofAAV. Compared with dystrophin, the modest size of DYSFimplores the development of novel ways to bypass pack-aging restraints with the benefit of restoring a full-lengthprotein. Three different approaches have been exploredfor generating full-length DYSF In Vivowith varying degreesof efficacy.54-56 Using a trans-splicing strategy, Lostal et al.56

engineered a two-vector system composed of one vectorcontaining the first half of DYSF and a splice donor site and asecond vector containing a splice acceptor site and the 30portion of DYSF. In DYSF-deficient mice, they demonstratedthat this approach was efficient and improved membranerepair by IM injection. However, when given systemically,expression was limited to <5% of muscle fibers.

We designed an alternative strategy taking advantage ofthe unique plasticity demonstrated by the AAV5 serotype.54

Using AAV5, we discovered that we could spontaneouslypackage two halves of DYSF yielding virions with regions ofhomology that result in a full-length transgene followingdelivery to muscle. Implementation of this approach leads torobust gene expression following direct IM or vasculardelivery using AAV5 to transfer DYSF to muscle (Fig 8). InDYSF-deficient muscle, the resulting expression improveshistology, protects the muscle membrane against laser-induced injury, and restores force and resistance to fatiguein the diaphragm. Long-term studies demonstrate sustainedDYSF expression for at least 1 year with no measurabledecrease in protein levels. Although only a clinical trial willbe able to define how these findings translate to treatment inLGMD2B patients, these preclinical data imply that deliveryof full-length DYSF by homologous recombination couldcorrect both the defects in membrane repair and the un-derlying histopathology of dysferlinopathy.

Conclusions

Remarkable progress has been made in gene therapy ap-proaches for muscular dystrophies with potential for trans-lation in clinical trials. This is the result of stepwisedevelopments providing a foundation on which to build.Success inexonskippingprovidesproof of concept fornonviralapproach to gene repair. Gene delivery using AAV demon-strates both safety and efficacy in local administration andwillbe further promoted through vascular delivery. Lessonslearned include careful screening of patients for pre-existingimmunity to either transgene or viral vector that will beused fordelivery.Alternate strategies thatusedisruptionof themyostatinpathwayalso reveal potentialmerit. An approach todeliver genes through homologous recombination asdemonstrated for DYSF may pay unexpected dividends inclinical trials. All in all, the future looks bright for multiplemolecular therapeutic strategies for themuscular dystrophies.

References

1. Klein CJ, Coovert DD, Bulman DE, Ray PN, Mendell JR, Burghes AHM.Somatic Reversion Suppression in Duchenne Muscular-Dystrophy(Dmd) - Evidence Supporting a Frame-Restoring Mechanism in RareDystrophin-Positive Fibers. Am J Hum Gen. 1992;50:950-959.

2. Aartsma-Rus A, Fokkema I, Verschuuren J, et al. Theoretic applica-bility of antisense-mediated exon skipping for Duchenne musculardystrophy mutations. Hum Mutat. 2009;30:293-299.

3. Mann CJ, Honeyman K, Cheng AJ, et al. Antisense-induced exonskipping and synthesis of dystrophin in the mdx mouse. Proc NatlAcad Sci U S A. 2001;98:42-47.

4. Goyenvalle A, Babbs A, Powell D, et al. Prevention of dystrophicpathology in severely affected dystrophin/utrophin-deficient miceby morpholino-oligomer-mediated exon-skipping. Mol Ther. 2010;18:198-205.

5. Yokota T, Lu QL, Partridge T, et al. Efficacy of systemic morpholinoexon-skipping in Duchenne dystrophy dogs. Ann Neurol. 2009;65:667-676.

6. van Deutekom JC, Janson AA, Ginjaar IB, et al. Local dystrophinrestoration with antisense oligonucleotide PRO051. N Engl J Med.2007;357:2677-2686.

7. Kinali M, Arechavala-Gomeza V, Feng L, et al. Local restoration ofdystrophin expression with the morpholino oligomer AVI-4658 inDuchenne muscular dystrophy: a single-blind, placebo-controlled,dose-escalation, proof-of-concept study. Lancet Neurol. 2009;8:918-928.

8. Goemans NM, Tulinius M, van den Akker JT, et al. Systemicadministration of PRO051 in Duchenne’s muscular dystrophy. NEngl J Med. 2011;364:1513-1522.

9. Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping anddystrophin restoration in patients with Duchenne muscular dys-trophy after systemic phosphorodiamidate morpholino oligomertreatment: an open-label, phase 2, dose-escalation study. Lancet.2011;378:595-605.

10. JR M, Rodino-Klapac LR, Sahenk Z, et al. Eteplirsen for the treatmentof Duchenne muscular dystrophy. Ann Neurol. 2013;74:637-647.

11. McDonald CM, Henricson EK, Abresch RT, et al. The 6-minutewalk testand other clinical endpoints in duchenne muscular dystrophy: Reli-ability, concurrent validity, and minimal clinically important differ-ences from a multicenter study.Muscle Nerve. 2013;48:357-368.

12. McDonald CM, Henricson EK, Han JJ, et al. The 6-minute walk test inDuchenne/Becker muscular dystrophy: longitudinal observations.Muscle Nerve. 2010;42:966-974.

13. Strober JB. Therapeutics in duchenne muscular dystrophy. NeuroRx.2006;3:225-234.

14. Mendell JR, Buzin CH, Feng J, et al. Diagnosis of Duchenne dystrophyby enhanced detection of small mutations. Neurology. 2001;57:645-650.

15. Flanigan KM, von Niederhausern A, Dunn DM, Alder J, Mendell JR,Weiss RB. Rapid direct sequence analysis of the dystrophin gene.Am J Hum Gen. 2003;72:931-939.

S. Al-Zaidy et al. / Pediatric Neurology 51 (2014) 607e618618

16. Du L, Damoiseaux R, Nahas S, et al. Nonaminoglycoside compoundsinduce readthrough of nonsense mutations. J Exp Med. 2009;206:2285-2297.

17. Du M, Jones JR, Lanier J, et al. Aminoglycoside suppression of apremature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. J Mol Med (Berl). 2002;80:595-604.

18. Barton-Davis ER, Cordier L, Shoturma DI, Leland SE, Sweeney HL.Aminoglycoside antibiotics restore dystrophin function to skeletalmuscles of mdx mice. J Clin Invest. 1999;104:375-381.

19. Wagner KR, Hamed S, Hadley DW, et al. Gentamicin treatment ofDuchenne and Becker muscular dystrophy due to nonsense muta-tions. Ann Neurol. 2001;49:706-711.

20. Politano L, Nigro G, Nigro V, et al. Gentamicin administration inDuchenne patients with premature stop codon. Preliminary results.Acta Myol. 2003;22:15-21.

21. Malik V, Rodino-Klapac LR, Viollet L, et al. Gentamicin-inducedreadthrough of stop codons in Duchenne muscular dystrophy. AnnNeurol. 2010;67:771-780.

22. Ahmad A, Brinson M, Hodges BL, Chamberlain JS, Amalfitano A. Mdxmice inducibly expressing dystrophin provide insights into thepotential of gene therapy for duchenne muscular dystrophy. HumMol Gen. 2000;9:2507-2515.

23. Ghahramani Seno MM, Graham IR, Athanasopoulos T, et al. RNAi-mediated knockdown of dystrophin expression in adult mice doesnot lead to overt muscular dystrophy pathology. Hum Mol Gen.2008;17:2622-2632.

24. Tang HY, Hutcheson E, Neill S, Drummond-Borg M, Speer M,Alford RL. Genetic susceptibility to aminoglycoside ototoxicity: howmany are at risk? Genet Med. 2002;4:336-345.

25. Viollet L, Gailey S, Thornton DJ, et al. Utility of Cystatin C to MonitorRenal Function in Duchenne Muscular Dystrophy. Muscle Nerve.2009;40:438-442.

26. Howard MT, Shirts BH, Petros LM, Flanigan KM, Gesteland RF,Atkins JF. Sequence specificity of aminoglycoside-induced stopcondon readthrough: potential implications for treatment ofDuchenne muscular dystrophy. Ann Neurol. 2000;48:164-169.

27. Welch EM, Barton ER, Zhuo J, et al. PTC124 targets genetic disorderscaused by nonsense mutations. Nature. 2007;447:87-91.

28. Hirawat S, Welch EM, Elfring GL, et al. Safety, tolerability, andpharmacokinetics of PTC124, a nonaminoglycoside nonsense mu-tation suppressor, following single- and multiple-dose administra-tion to healthy male and female adult volunteers. J Clin Pharmacol.2007;47:430-444.

29. Quinlivan R KJ, Comi G, Bushby K, the Ataluren DBMD Study Group.Safety and efficacy of ataluren 10,10,20 mg/kg TID in patients withnonsense mutation dystrophinopathy. European PaediatricNeurology Society Congress, 9th EPNS Congress [online]; 2010.

30. Mendell JR, Moxley RT, Griggs RC, et al. Randomized, double-blindsix-month trial of prednisone in Duchenne’s muscular dystrophy. NEngl J Med. 1989;320:1592-1597.

31. Griggs RC, Herr BE, Reha A, et al. Corticosteroids in Duchennemuscular dystrophy: major variations in practice. Muscle & Nerve.2013;48:27-31.

32. Mendell JR, Shilling C, Leslie ND, et al. Evidence-based path tonewborn screening for Duchenne muscular dystrophy. Ann Neurol.2012;71:304-313.

33. Mendell JR, Lloyd-Puryear M. Report of MDA muscle disease sym-posium on newborn screening for Duchenne muscular dystrophy.Muscle & Nerve. 2013;48:21-26.

34. England SB, Nicholson LV, Johnson MA, et al. Very mild musculardystrophy associated with the deletion of 46% of dystrophin. Nature.1990;343:180-182.

35. Wang B, Li J, Xiao X. Adeno-associated virus vector carrying humanminidystrophin genes effectively ameliorates muscular dystrophyin mdx mouse model. Proc Natl Acad Sci U S A. 2000;97:13714-13719.

36. Watchko J, O’Day T, Wang B, et al. Adeno-associated virus vector-mediated minidystrophin gene therapy improves dystrophic musclecontractile function inmdxmice.HumGene Ther. 2002;13:1451-1460.

37. Mendell JR, Campbell K, Rodino-Klapac L, et al. Dystrophin immu-nity in Duchenne’s muscular dystrophy. N Engl J Med. 2010;363:1429-1437.

38. Flanigan KM, Campbell K, Viollet L, et al. Anti-Dystrophin T CellResponses in Duchenne Muscular Dystrophy: Prevalence and aGlucocorticoid Treatment Effect. Hum Gene Ther Methods. 2013;24:797-806.

39. Moore S, Shilling C, Mendell J. Limb-Girdle Muscular Dystrophy inthe United States. J Neuropath Exp Neurol. 2006;65:995-1003.

40. Rodino-Klapac LR, Lee JS, Mulligan RC, Clark KR, Mendell JR. Lack oftoxicity of alpha-sarcoglycan overexpression supports clinical genetransfer trial in LGMD2D. Neurology. 2008;71:240-247.

41. Mendell JR, Rodino-Klapac LR, Rosales-Quintero X, et al. Limb-girdlemuscular dystrophy type 2D gene therapy restores alpha-sarco-glycan and associated proteins. Ann Neurol. 2009;66:290-297.

42. Mendell JR, Rodino-Klapac LR, Rosales XQ, et al. Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdlemuscular dystrophy, type 2D. Ann Neurol. 2010;68:629-638.

43. Toromanoff A, Cherel Y, Guilbaud M, et al. Safety and efficacy ofregional intravenous (r.i.) versus intramuscular (i.m.) delivery ofrAAV1 and rAAV8 to nonhuman primate skeletal muscle. Mol Ther.2008;16:1291-1299.

44. Toromanoff A, Adjali O, Larcher T, et al. Lack of immunotoxicity afterregional intravenous (RI) delivery of rAAV to nonhuman primateskeletal muscle. Mol Ther. 2010;18:151-160.

45. Rodino-Klapac LR, Montgomery CL, Bremer WG, et al. Persistentexpression of FLAG-tagged micro dystrophin in nonhuman pri-mates following intramuscular and vascular delivery. Mol Ther.2010;18:109-117.

46. Hegge JO, Wooddell CI, Zhang G, et al. Evaluation of hydrodynamiclimb vein injections in nonhuman primates. Hum Gene Ther. 2010;21:829-842.

47. Arruda VR, Stedman HH, Nichols TC, et al. Regional intravasculardelivery of AAV-2-F.IX to skeletal muscle achieves long-termcorrection of hemophilia B in a large animal model. Blood. 2005;105:3458-3464.

48. Katwal AB, Konkalmatt PR, Piras BA, et al. Adeno-associated virusserotype 9 efficiently targets ischemic skeletal muscle followingsystemic delivery. Gene Ther. 2013;20:930-938.

49. Rodino-Klapac LR, Montgomery CL, Mendell JR, Chicoine LG. AAV-mediated gene therapy to the isolated limb in rhesus macaques.Methods Mol Biol. 2011;709:287-298.

50. Rodino-Klapac LR, Janssen PM, Montgomery CL, et al. A translationalapproach for limb vascular delivery of the micro-dystrophin genewithout high volume or high pressure for treatment of Duchennemuscular dystrophy. J Transl Med. 2007;5:45.

51. Liu J, Aoki M, Illa I, et al. Dysferlin, a novel skeletal muscle gene, ismutated in Miyoshi myopathy and limb girdle muscular dystrophy.Nature Genetics. 1998;20:31-36.

52. Illa I, Serrano-Munuera C, Gallardo E, et al. Distal anteriorcompartment myopathy: a dysferlin mutation causing a newmuscular dystrophy phenotype. Ann Neurol. 2001;49:130-134.

53. Nagashima T, Chuma T, Mano Y, et al. Dysferlinopathy associatedwith rigid spine syndrome. Neuropathology. 2004;24:341-346.

54. Grose WE, Clark KR, Griffin D, et al. Homologous RecombinationMediates Functional Recovery ofDysferlinDeficiency followingAAV5Gene Transfer. Plos One. 2012;7.

55. Krahn M, Wein N, Bartoli M, et al. A naturally occurring humanminidysferlin protein repairs sarcolemmal lesions in a mouse modelof dysferlinopathy. Sci Transl Med. 2010;2:50ra69.

56. Lostal W, Bartoli M, Bourg N, et al. Efficient recovery of dysferlindeficiency by dual adeno-associated vector-mediated gene transfer.Hum Mol Gen. 2010;19:1897-1907.