Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy

Dual AAV therapy ameliorates exercise-inducedmuscle injury and functional ischemia in murinemodels of Duchenne muscular dystrophy

Yadong Zhang1,{, Yongping Yue1, Liang Li2, Chady H. Hakim1, Keqing Zhang1,

Gail D. Thomas2 and Dongsheng Duan1,∗

1Department of Molecular Microbiology and Immunology, The University of Missouri, Columbia, MO, USA2The Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA

Received January 25, 2013; Revised April 6, 2013; Accepted May 13, 2013

Neuronal nitric oxide synthase (nNOS) membrane delocalization contributes to the pathogenesis of Duchennemuscular dystrophy (DMD) by promoting functional muscle ischemia and exacerbating muscle injury duringexercise. We have previously shown that supra-physiological expression of nNOS-binding mini-dystrophinrestores normal blood flow regulation and prevents functional ischemia in transgenic mdx mice, a DMDmodel. A critical next issue is whether systemic dual adeno-associated virus (AAV) gene therapy can restorenNOS-binding mini-dystrophin expression and mitigate muscle activity-related functional ischemia andinjury. Here, we performed systemic gene transfer in mdx and mdx4cv mice using a pair of dual AAV vectorsthat expressed a 6 kb nNOS-binding mini-dystrophin gene. Vectors were packaged in tyrosine mutant AAV-9and co-injected (5 3 1012 viral genome particles/vector/mouse) via the tail vein to 1-month-old dystrophin-nullmice. Four months later, we observed 30–50% mini-dystrophin positive myofibers in limb muscles.Treatment ameliorated histopathology, increased muscle force and protected against eccentric contraction-induced injury. Importantly, dual AAV therapy successfully prevented chronic exercise-induced muscle forcedrop. Doppler hemodynamic assay further showed that therapy attenuated adrenergic vasoconstriction in con-tracting muscle. Our results suggest that partial transduction can still ameliorate nNOS delocalization-asso-ciated functional deficiency. Further evaluation of nNOS binding mini-dystrophin dual AAV vectors iswarranted in dystrophic dogs and eventually in human patients.

INTRODUCTION

Dystrophin deficiency results in Duchenne muscular dystrophy(DMD), the most common lethal inherited muscle disease inboys (1). The 2.4 mb dystrophin gene contains 79 exons and ittranscribes into a 14 kb cDNA. A highly promising approachto treat DMD is to restore dystrophin expression in all musclecells in the body using gene replacement therapy. Currently,adeno-associated virus (AAV) is the only vector with proven evi-dence of whole body muscle transduction in small (such as mice)and large (such as dogs) animals (2,3). However, AAV is thesmallest DNA virus with a viral particle size of only 20–25 nm. The maximal packaging capacity of an AAV vector is

�5 kb (4). This is far below the size of the full-length dystrophincDNA (�12 kb).

To overcome this hurdle, investigators have tested a variety ofsmaller quasi-functional dystrophin genes. Among these, thenaturally occurring D17-48 mini-dystrophin gene and the syn-thetic DH2-R19 mini-dystrophin gene are extremely promising.The D17-48 minigene was isolated from a 60-year-old mildlyaffected patient (5). This patient carried a large in-frame deletionwhich removed 46% of the dystrophin gene spanning from exon17 to 48. The DH2-R19 minigene is an improved version of theD17-48 minigene (6). Compared with the D17-48 minigene, theDH2-R19 minigene is more effective in preventing muscledegeneration and recovering muscle force (6).

†Present address: The Central Hospital of Wuhan, Wuhan, Hubei 430014, China.

∗To whom correspondence should be addressed at: Department of Molecular Microbiology and Immunology, The University of Missouri, School ofMedicine, One Hospital Dr, M610G, MSB, Columbia, MO 65212, USA. Tel: +1 5738849584; Fax: +1 5738824287; Email: [email protected]

# The Author 2013. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2013, Vol. 22, No. 18 3720–3729doi:10.1093/hmg/ddt224Advance Access published on May 15, 2013

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An essential function of dystrophin is to localize neuronalnitric oxide synthase (nNOS) to the sarcolemma (7).Membrane-associated nNOS is required to optimize muscle per-fusion during contraction by protecting against excessive adren-ergic vasoconstriction (8–10). Loss of sarcolemmal nNOS leadsto muscle ischemia and accelerates the dystrophic process inDMD (8,10–12). Unfortunately, DH2-R19 mini-dystrophincannot anchor nNOS to the muscle cell membrane (13,14). Werecently found that dystrophin spectrin-like repeats 16 and 17(R16/17) is the nNOS-binding domain (8,15). Importantly, thenNOS-binding domain R16/17 is absent in the DH2-R19 mini-gene (6). To increase the biological activity of the DH2-R19minigene, we engineered R16/17 into mini-dystrophin and gen-erated the nNOS-binding domain containing minigene (8,16).Transgenic over-expression of this minigene successfully atte-nuated functional ischemia and muscle injury in mdx mice, acommonly used mouse DMD model (8). Based on this encour-aging finding, we developed a series of novel dual AAVvectors to express the nNOS-binding minigene (16). In a prelim-inary local muscle injection study, we found that one of our dualAAV sets (YZ27 and YZ22) resulted in robust mini-dystrophinexpression in dystrophin-null mdx4cv mice and successfullyestablished sarcolemmal nNOS expression (Fig. 1A) (16). Inthe current study, we examined whether systemic co-deliveryof the YZ27 and YZ22 dual AAV vectors can restore normalblood flow regulation in contracting muscle and preventexercise-induced muscle injury.

RESULTS

Systemic delivery of the mini-dystrophin dual AAVvectors results in broad skeletal muscle transductionin young adult dystrophic mice

We recently engineered a pair of dual AAV vectors (YZ27 andYZ22) to express the nNOS-binding DR2-15/DR18-19 mini-dystrophin gene (Fig. 1A) (16). This minigene carries the R16/17 nNOS-binding domain. For easy detection of the full-lengthprotein, a GFP gene and a flag-tag were fused to the N-terminaland C-terminal end, respectively. Initial characterization byYZ27/YZ22 co-infection in the anterior tibialis muscle ofdystrophin-deficient mice confirmed in-frame GFP expressionand nNOS binding (16). Injection of either YZ22 alone orYZ27 alone did not yield GFP or dystrophin fragment expression(data not shown) (16). To determine whether intravenous deliv-ery can lead to bodywide muscle transduction, we generatedY731F tyrosine mutant AAV-9 dual vectors. Dual vectorswere co-delivered via the tail vein at the dose of 5 × 1012 vg par-ticles/vector/mouse to 1-month-old mdx4cv mice, a strain ofdystrophin-null mice that have been used in dual AAV mini-dystrophin gene therapy studies before (16,17). Four monthsafter gene transfer, we evaluated transduction efficiency(Fig. 1). Consistent with our previous study (16), allGFP-positive myofibers were also positive for mini-dystrophin,Flag tag and nNOS (Fig. 2 and data not shown). Considerable ex-pression was observed in the upper limb, extensor digitorum

Figure 1. Co-delivery of the mini-dystrophin dual AAV vectors through the vasculature results in efficient limb and abdominal muscle transduction. (A) The sche-matic outline of the DR2-15/DR18-19 mini-dystrophin dual AAV vectors. YZ27 carries the 5′ part of the minigene. YZ22 carries the 3′ part of the minigene. A GFPgene is fused in-frame to the N-terminal end of the mini-dystrophin gene. The nNOS-binding domain is located in dystrophin repeats 16 and 17. CMV, the cytomegalo-virus promoter; pA, the polyadenylation signal; Hairpin-like structure, inverted terminal repeats of AAV virus; N, the N-terminal domain of dystrophin; H1, H3 andH4, hinges 1, 3 and 4 in the dystrophin rod domain; Numerical numbers, spectrin-like repeats in the dystrophin rod domain. (B) Representative direct fluorescencephotomicrographs revealing GFP expression in different skeletal muscles. UL, upper limb muscle; EDL, extensor digitorum longus muscle; TA, tibialis anteriormuscle; Quad, quadriceps muscle; Gas, gastrocnemius muscle; Abd, abdominal muscle. Scale bar: 100 mm. (C) Quantitative evaluation of GFP-positive myofibersin the indicated muscles. Results are from nine-infected mdx4cv mice (mean+SEM). (D) Representative western blot results from indicated muscles. Top panel isprobed with an anti-dystrophin C-terminal antibody. This antibody recognizes both full-length dystrophin in wild-type (WT) mice and mini-dystrophin expressedfrom the dual AAV vectors. Open arrowhead, full-length dystrophin; filled arrowhead, DR2-15/DR18-19 mini-dystrophin. Bottom panel is probed with theGFP antibody.

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longus (EDL), tibialis anterior (TA), quadriceps, gastrocnemiusand abdominal muscles (Fig. 1B). Quantification of the positivemyofibers showed that �30–50% of myofibers expressed mini-dystrophin in these muscles (Fig. 1C). Limb muscle western blot

revealed a single band corresponding to the predicted size of theGFP-fused mini-dystrophin protein (Fig. 1D). Consistent with aprevious study, limited expression was observed in the heart anddiaphragm (data not shown) (17).

Figure 2. Dual AAV-mediated mini-dystrophin expression ameliorates histological lesions in dystrophic muscle. (A) Representative photomicrographs of the quad-riceps muscle from BL6, mdx4cv and dual AAV-infected mdx4cv mice revealing dystrophin expression, general histology (HE) and nNOS expression. GFP, directfluorescence photomicrographs; Hum Dys, indirect immunofluorescence staining with an antibody specific for human dystrophin; R4-6, indirect immunofluorescencestaining with an antibody against dystrophin spectrin-like repeats 4 to 6. This region is deleted in theDR2-15/DR18-19 mini-dystrophin gene; nNOS activity, enzym-atic staining for nNOS activity; nNOS IF, indirect immunofluorescence staining with an antibody against nNOS. Asterisk marks the same myofiber in serial sections.Black/white square, a myofiber not transduced by mini-dystrophin dual AAV vectors. (B) Quantification of centrally nucleated myofibers (CN) in the tibialis anteriormuscle (TA) and the gastrocnemius muscle (Gastro). Sample size (number of muscles studied): N ¼ 4 for BL6, N ¼ 5 for mdx4cv, N ¼ 9 muscles for AAV-infectedmdx4cv. Approximately 100–250 myofibers were quantified for each indicated category. AAV neg myofiber, untransduced myofibers in dual AAV-infected mdx4cvmice; AAV pos myofiber, mini-dystrophin positive myofibers in dual AAV-infected mdx4cv mice. Asterisk, significantly different from that of BL6 and AAV posmyofiber. (C) Distribution of myofiber size in normal, mdx4cv and AAV-treated mdx4cv mice. Top panel, TA muscle; bottom panel, gastrocnemius muscle.Approximately 680 myofibers were measured for each muscle in each strain.

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Dual AAV gene transfer restores sarcolemmal nNOSexpression and improves muscle histopathologyin mdx4cv muscle

To validate dual vector-mediated DR2-15/DR18-19 mini-dystrophin expression, serial muscle sections were examinedfor GFP, dystrophin and nNOS expression (Fig. 2A). Asexpected, GFP-positive myofibers were only observed in dualAAV-infected mdx4cv muscle. In adjacent sections, myofibersthat were positive for GFP were recognized by the human dys-trophin specific antibody (Hum Dys) but not by the R4-6 anti-body, an antibody that reacts with an epitope absent in thesynthetic mini-dystrophin gene (Fig. 2A). To evaluatemembrane-bound nNOS expression, we performed nNOS im-munofluorescence staining and in situ nNOS enzymatic activityassay. Both methods confirmed correct localization of nNOS onthe muscle cell membrane in dual AAV-treated mdx4cv muscle(Fig. 2A).

Hematoxylin and eosin (HE) staining revealed histology im-provement. In dystrophic muscles that received intravenousdual AAV injection, there appeared to be less inflammation, de-generation/regeneration and necrosis (Fig. 2A). Quantificationof the percentage of centrally nucleated myofiber and the distri-bution of myofiber size further confirmed HE staining results(Fig. 2B and C). In both the TA and gastrocnemius muscles,central nucleation was significantly reduced in myofibers thatwere transduced by the dual vectors (AAV mini-dystrophin posi-tive myofibers) but was not altered in myofibers that were nottransduced (AAV mini-dystrophin negative myofibers)(Fig. 2B). Next, we examined myofiber size distribution in theTA and gastrocnemius muscles (Fig. 2C). Compared withnormal C57Bl/6 (BL6) muscle, mdx4cv muscle showed amuch broader range with more very small and very large myofi-bers. Dual AAV treatment shifted the pattern of myofiber sizedistribution. It was not completely normalized but was clearlyimproved compared with that of non-injected mdx4cv mice(Fig. 2C).

Intravenous administration of the mini-dystrophindual AAV vectors increases muscle function in theDMD mouse model

To fully evaluate muscle physiology, we conducted ex vivo andin situ force measurement in the EDL and TA muscle, respect-ively (Fig. 3A–C). The physiological properties of the EDLmuscle were studied in Ringer’s buffer using freshly isolatedmuscle (Fig. 3). Compared with that of untreated mdx4cvmice, the dual AAV-treated mdx4cv EDL muscle had a signifi-cantly smaller muscle mass and cross-sectional area (CSA).Mini-dystrophin gene therapy significantly increased specifictetanic force of the EDL muscle. Upon repeated cycles of eccen-tric contraction stress, AAV-treated EDL muscle showed signifi-cantly less force loss (Fig. 3C). TA muscle force was measured inthe intact animal in situ (Fig. 3B). Similar to what we saw in theEDL muscle, dual AAV therapy also significantly reducedmuscle weight and CSA in the TA muscle (Fig. 3B). Consistent-ly, treatment significantly enhanced tetanic forces of the TAmuscle (Fig. 3B).

Dual vector-mediated mini-dystrophin expression preventsexercise-induced loss of muscle force and amelioratesfunctional muscle ischemia in dystrophin-deficient mice

Two different approaches were used to determine whether dualAAV-mediated DR2-15/DR18-19 mini-dystrophin expressioncan treat the nNOS-dependent phenotypes of exercise-inducedmuscle force reduction and functional muscle ischemia. First,we examined muscle force change following chronic exercisechallenge in mdx4cv mice. This method has been previouslyused to demonstrate focal muscle injury and compromisedmuscle force production in Fiona mice, a strain of full-lengthutrophin transgenic mdx mice (18). In this assay, mice wererun on a treadmill twice a week for a total of eight weeks. Atthe end of the treadmill sessions, the EDL muscle force was mea-sured and compared with that of non-exercised mice. In normalBL6 mice, repeated treadmill running did not compromise forceproduction in the EDL muscle (Fig. 3D). In untreated mdx4cvmice, chronic treadmill challenge resulted in a significant reduc-tion in the EDL muscle specific force at the stimulation fre-quency of 50, 100 and 150 Hz (a strong trend of reduction wasseen at 80 Hz, but it did not reach statistical significance)(Fig. 3E). In sharp contrast, chronic treadmill exercise had anominal effect on specific muscle force in dual AAV-treatedmdx4cv mice (Fig. 3F). In other words, restoration of sarcolem-mal nNOS by dual AAV-mediated mini-dystrophin therapy hassuccessfully prevented muscle damage and the subsequent lossof force in mdx4cv mice.

To determine whether we can achieve the same transductionefficiency in a different strain of dystrophin-null mice, we per-formed tail vein injection in 1-month-old mdx mice at thesame dose as we have used for mdx4cv mice (5 × 1012 vg parti-cles/vector/mouse) (Fig. 4 and Supplementary Material,Fig. S1). Consistent with our findings in mdx4cv mice (Fig. 1),intravenous dual AAV administration resulted in similar trans-duction efficiency in mdx mice (Fig. 4A and Supplementary Ma-terial, Fig. S1). As exemplified in the TA muscle, �30–50% ofmyofibers were transduced (Fig. 4A). All GFP-positive myofi-bers displayed sarcolemmal nNOS expression (Fig. 4B).

We have previously used an in vivo hemodynamic assay toshow that mdx mice are susceptible to muscle ischemia duringexercise due to loss of sarcolemmal nNOS (8,10). In thisassay, we measured the transient decrease in hindlimb bloodflow caused by femoral artery injection of the sympathetic vaso-constrictor norepinephrine when the hindlimb muscles were atrest. We then stimulated the sciatic nerve to evoke hindlimbmuscle contractions and repeated the norepinephrine injection.Norepinephrine-mediated vasoconstriction normally is attenu-ated in the contracting hindlimbs in normal C57Bl/10 (BL10)mice, which optimizes blood flow to the working muscles(Fig. 4C) (8,10). Consistent with our previous publications(8,10), norepinephrine evoked similar decreases in femoral vas-cular conductance in the resting and contracting hindlimbmuscles in mdx mice, directly demonstrating functionalmuscle ischemia (Fig. 4C). Following mini-dystrophin dualAAV treatment, adrenergic vasoconstriction was significantlyattenuated during muscle contraction, showing a clear treatmenteffect in ameliorating functional muscle ischemia (Fig. 4C).




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Figure 3. Dual AAV minigene therapy corrects muscle hypertrophy, enhances contractility and preserves force generation when challenged with intensive chronictreadmill exercise. (A) Comparison of the EDL muscle weight, CSA, specific twitch and tetanic tension among BL6, untreated mdx4cv and dual AAV-treated mdx4cvmice. (B) Comparison of the TA muscle weight, CSA, specific twitch and tetanic tension among BL6, untreated mdx4cv and dual AAV-treated mdx4cv mice.(C) Eccentric contraction profiles of the EDL muscle in BL6, treated and untreated mdx4cv mice. (A–C) Asterisk indicates that the value in dual AAV-treatedmdx4cv muscle is significantly different from those of untreated mdx4cv and BL6 muscles. (A and B) Cross indicates that the value in dual AAV-treated mdx4cvmuscle is significantly different from that of untreated mdx4cv muscle only. (D) Quantitative comparison of specific muscle force of the EDL muscle in BL6mice with or without chronic treadmill running. (E) Quantitative comparison of specific muscle force of the EDL muscle in mdx4cv mice with or without chronictreadmill running. Asterisk, significantly higher than treadmill challenged mice. (F) Quantitative comparison of specific muscle force of the EDL muscle in dualAAV-treated mdx4cv mice with or without chronic treadmill running.




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DISCUSSION

In this study, we evaluated therapeutic benefits of the nNOS-recruiting dual AAV vectors in mouse models of DMD by sys-temic gene transfer. We demonstrated that intravascularco-injection of two independent AAV vectors successfullyreconstituted an intact mini-dystrophin expression cassette inthe majority of muscles of young adult dystrophic mice(Figs 1, 4 and Supplementary Material, Fig. S1). With an encour-aging transduction efficiency (�30–50% of limb muscle myo-fiber were positive for mini-dystrophin), dual AAV therapysignificantly ameliorated dystrophic pathology and improvedmuscle function (Figs 2 and 3). Specifically, treated miceshowed less muscle inflammation, a significant reduction ofmyofiber degeneration/regeneration (as reflected by centralnuclear quantification) and a partial correction of abnormal myo-fiber size distribution (Fig. 2). Dual AAV therapy also signifi-cantly alleviated muscle pseudohypertrophy, a uniquedisease-related feature in young adult dystrophic mice (Fig. 3)(6,19,20). Physiology assays demonstrated that treatment sig-nificantly enhanced tetanic muscle force and preserved musclecontractility under eccentric contraction stress (Fig. 3). Most im-portantly, dual AAV therapy reestablished sarcolemmal nNOSdistribution in dystrophic muscle (Figs 2 and 4). As a conse-quence, norepinephrine-induced muscle ischemia during exer-cise was significantly blunted and muscle force loss caused byrepeated exercise was prevented (Figs 3 and 4).

Functional muscle ischemia caused by nNOS delocalizationhas been recognized as a critical pathogenic mechanism in

DMD for more than a decade (7,8,10,11). It may underlie anumber of characteristic symptoms in patients such as musclecramp, fatigue and force reduction. For this reason, treatingmuscle ischemia is an important goal in DMD gene therapy.Unfortunately, none of the gene therapy studies published sofar has accomplished this mission (21). The molecular mechan-ism underlying dystrophic functional muscle ischemia has beendelineated (10). In normal muscle, nitric oxide (NO) producedby sarcolemmal nNOS attenuates a-adrenergic vasoconstric-tion, thereby optimizing blood flow to meet the metabolicneeds of the contracting muscles. Correct position of nNOS atthe sarcolemma is vital in this process in order to facilitateready diffusion of NO to the adjacent vasculature. In dystrophin-deficient muscle, nNOS is lost from the sarcolemma and the totalnNOS content is reduced (7). As a result, NO-mediated vasodila-tion is compromised, resulting in functional muscle ischemia.Re-directing nNOS back to the membrane should restoreNO-mediated blood flow regulation in contracting muscle andeliminate functional ischemia.

Early studies suggested that nNOS is connected to the sarco-lemma by syntrophin (22). For this reason, it seems that restor-ation of syntrophin to the muscle cell membrane would besufficient to localize nNOS to the sarcolemma. In support ofthis theory, Wang et al. (23) claimed that they achievedmembrane-associated nNOS expression using a minimized dys-trophin construct containing repeats R1, R2, R22, R23 and R24.Surprisingly, similar mini-/micro-dystrophin genes used byother laboratories have all failed to bring nNOS to the

Figure 4. Restoration of sarcolemmal nNOS expression by dual AAV minigene therapy ameliorates functional muscle ischemia. (A) Representative low magnifica-tion photomicrographs of nNOS activity staining of the TA muscle from a dual AAV-treated mdx mouse. High magnification images of the boxed areas are shown inthe right column of (B). (B) Representative high power photomicrographs of the TA muscle from untreated (left column) and dual AAV-treated (right column) mdxmice. Top panel, GFP expression; middle panel, nNOS immunofluorescence staining; bottom panel, in situ nNOS enzymatic activity staining. Asterisk, the samemyofiber in serial sections. (C) Quantitative comparison of the femoral vascular conductance response to norepinephrine in resting and contracting hindlimbs ofBL10, untreated mdx and dual AAV-treated mdx mice. Asterisk, significantly different between resting and contracting muscle.




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membrane, although syntrophin is recruited back to the sarco-lemma by these minimized genes (8,13,14,24). We recentlyfound that in addition to syntrophin, membrane targeting ofnNOS also requires an nNOS-binding domain located in dys-trophin R16/17 (8,15,18). Synthetic dystrophin genes thatcarry this domain can successfully restore nNOS to the mem-brane, while constructs that do not contain R16/17 cannot.

To translate our findings to DMD therapy, we decided to en-gineer the R16/17 nNOS recruiting domain to the DH2-R19mini-dystrophin gene. We chose the DH2-R19 minigenebecause this minigene is originated from a truncated dystrophingene in an extremely mild patient (5). Among dozens of abbre-viated synthetic dystrophin constructs, the DH2-R19 minigeneis the only one that we know works in human muscle (21). In apreliminary study, we compared two strains of transgenic mdxmice that either expressed the DH2-R19 minigene or ournewly engineered nNOS-binding minigene (8). Encouragingly,supra-physiological expression of the nNOS-binding minigene,but not the DH2-R19 minigene, effectively normalized muscleblood flow regulation and prevented ischemic injury duringexercise in transgenic mdx mice (8). In summary, our prelimin-ary study has clearly established the therapeutic advantage of thenNOS-binding minigene and supports preclinical testing of thenNOS-binding minigene by AAV-mediated gene therapy.

The nNOS-binding minigene (DR2-15/DR18-19) is 6 kb. Thisexceeds the 5 kb packaging limit of a single AAV virion (4). Inorder to deliver a large gene with AAV, we have developed anumber of dual vector strategies such as trans-activation, trans-splicing,overlappingandhybridsystems(13,16,25–28).Thefun-damental idea behind these approaches is to divide a large geneinto two parts and package them in two different AAV vectors.The intact gene is reconstituted at the DNA level after co-infectionand inter-molecular recombination of two different AAVgenomes (29,30). Subsequent studies from several laboratories,including ours, demonstrate that dual AAV vectors not onlyresult in saturated expression after direct muscle injection butalso yield whole body muscle transduction following intravascu-lar delivery (17,31–33). Collectively, these results reveal a highfeasibility of treating muscular dystrophy with dual AAV vectors.

In light of abundant literature support, we developed the YZ27/YZ22 dual AAV vector set to express the 6 kb nNOS-bindingmini-dystrophin gene (16). This set of vectors is designed usingthe principle of the overlapping strategy (27,34). Basically, thetail part of the 5′-end gene fragment is identical to that of thehead part of the 3′-end gene fragment. Reconstitution is achievedthrough homologous recombination. Following systemicco-injection of the YZ27 and YZ22 vectors, mini-dystrophin ex-pression was observed in 30–50% of the myofibers in all limbmuscles and some body wall muscles (such as the abdominalmuscle) (Figs 1 and 4). This level of mosaic expression is pre-dicted to be therapeutically relevant in terms of reducing musclepathologyandincreasingmusclestrength(35). Indeed,histologic-al examination and force measurement showed significant im-provement in treated mice (Figs 2 and 3).

The primary goal of the current study is to test whether recon-stitution of membrane-associated nNOS in a subset of myofiberscan attenuate functional muscle ischemia in dystrophic mice.This is a highly relevant question because gene therapy is unlike-ly going to yield supra-physiological expression in every myofi-ber as we have seen in transgenic mice (8). To determine whether

partial transduction (�30–50% mini-dystrophin positive myo-fibers) can ameliorate functional muscle ischemia and reducemuscle injury, we have taken two independent approachesusing two different strains of dystrophin-null mice (mdx andmdx4cv) (Figs 3 and 4). Mdx4cv mice are on the BL6 back-ground and they carry a nonsense mutation in exon 50 (36).Mdx mice are naturally occurring dystrophin-null mice on theBL10 background and their mutation is located in exon 23(37). For mdx4cv mice, we challenged them with chronic tread-mill exercise. In this assay, the hindlimb EDL muscle generatesmuch less force due to repeated muscle injury in the absence ofsarcolemmal nNOS (18). Encouragingly, treadmill running didnot result in EDL force loss in dual AAV-treated mdx4cvmice, suggesting that the restoration of nNOS has protectedagainst exercise-induced muscle damage (Fig. 3F). We thenused mdx mice to perform complementary experiments to dir-ectly quantify hindlimb blood flow regulation. The mdx mouseis the model originally used to establish the concept of functionalischemia in exercising dystrophic muscle (10). Consistent withthe findings in mdx4cv mice (Fig. 3F), AAV treatment restoredsarcolemmal nNOS and significantly reduced norepinephrine-mediated ischemia in contracting mdx muscle (Fig. 4C). Never-theless, the protection offered by the nNOS-binding dual AAVtreatment in this study (�30% less hindlimb ischemia) wasnot as robust as the protection we previously observed in BL10mice and transgenic mdx mice which had supra-physiologic ex-pression of the nNOS-binding mini-dystrophin gene (�80% lesshindlimb ischemia) (Fig. 4C) (8,10). Taken together, the resultsfrom two complementary models/assays confirm that our dualAAV therapy has attenuated functional muscle ischemiaduring exercise and prevented muscle injury and the loss ofmuscle force induced by repeated exercise in the murine DMDmodel. This is an important milestone since it demonstrates forthe first time that gene therapy can alleviate known functionalconsequences of nNOS membrane delocalization such as func-tional muscle ischemia, muscle injury and compromised forcegeneration. On the other hand, our studies also suggest that add-itional optimization is needed to enhance transduction efficiencyand achieve even better muscle protection.

MATERIALS AND METHODS

Animals

Animal experiments were approved by the Animal Care and UseCommittees of the University of Missouri and Cedars-SinaiMedical Center and were in accordance with NIH guidelines.BL6, BL10, mdx4cv (B6Ros.Cg-Dmdmdx-4Cv/J) and mdx(C57BL/10ScSn-Dmdmdx/J) mice were purchased from TheJackson Laboratory (Bar Harbor, ME, USA). Only young adultmale mice were used in the study. All mice were housed inspecific-pathogen free animal care facilities and kept under a12 h light (25 lux)/12 h dark cycle with free access to food andwater.

AAV production and gene delivery

The proviral cis plasmids have been published before (16). Theyinclude YZ27 and YZ22 (Fig. 1A). YZ27 contains the cyto-megalovirus (CMV) promoter, a GFP gene fused to the




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N-terminal end of the dystrophin gene, and the 5′-end of theDR2-15/DR18-19 mini-dystrophin gene including the N-terminal domain, hinges 1 and 3, and spectrin-like repeats R1,R16, R17, R20 and a part of R21. YZ22 contains the 3′-end ofthe DR2-15/DR18-19 mini-dystrophin gene including a part ofhinge 3, spectrin-like repeats R20 to R24, hinge 4, cysteine-richdomain and the C-terminal domain. YZ22 also contains a flag tagand the SV40 poly-adenylation signal. The Y731F tyrosinemodified AAV-9 was purified and titrated according to our pub-lished protocol using a cap/rep helper plasmid generously pro-vided by Dr Arun Srivastava (University of Florida,Gainesville, FL, USA) (38,39). Dual AAV vectors were deliv-ered in pairs to 1-month-old mdx or mdx4cv mice via the tailvein. Each co-infection consisted of 5 × 1012 viral genome(vg) particles/vector/mouse. AAV transduction was evaluatedat 4 months post-injection.

Morphology studies

General histology was examined by HE staining. GFP was visua-lized under the FITC channel using a Nikon E800 fluorescencemicroscope. Photomicrographs were taken with a QimageREtiga 1300 camera. The DR2-15/DR18-19 mini-dystrophinwas examined with Dys 3, a human dystrophin-specific mousemonoclonal antibody against an epitope located in dystrophinhinge 1 (1:20; Novocastra, Newcastle, UK) (8,13,40). Endogen-ous mouse dystrophin was detected with the H-300 rabbit poly-clonal antibody against an epitope located between R4 to R6(1:400; Santa Cruz Biotechnology, Santa Cruz, CA, USA).The nNOS protein was detected with a polyclonal antibody(1:2000; Santa Cruz Biotechnology). In situ NOS activity stain-ing was performed according to our published protocol(8,12,18). The percentage of centrally nucleated myofiberswas determined by manually counting the total number of myo-fibers and the total number of myofibers carrying centrallylocated nuclei in 8 mm HE stained sections. The percentage ofcentral nucleation was calculated with the formula, % centralnucleation ¼ 100 × (total number of myofibers carryingcentrally located nuclei)/(total number of myofibers). In mini-dystrophin dual AAV vector infected mdx4cv mice, the percent-age of centrally nucleated myofibers was determined separatelyfor mini-dystrophin positive and mini-dystrophin negative myo-fibers. In these cases, mini-dystrophin expression was confirmedin the adjacent section. The myofiber size was determined fromthe digitized images using the quantitative image analysismodule of the extended version of the Photoshop CS5.5 software(Adobe Systems Incorporated, San Jose, CA, USA).

Western blot

Whole muscle lysate was obtained from frozen limb musclesaccording to our previous publications (12,16). Dystrophinwas detected with a monoclonal antibody against the dystrophinC-terminal domain (Dys2, 1:100, clone Dy8/6C5, IgG1;Novocastra, Newcastle, UK). This antibody recognizes bothendogenous full-length dystrophin and DR2-15/DR18-19 mini-dystrophin. GFP was determined with a monoclonal antibodyagainst GFP (1:2000, clone 3E6, IgG2a; Invitrogen, Carlsbad,CA, USA).

Ex vivo evaluation of the EDL muscle function

Mice were anesthetized via intra-peritoneal injection of a cocktailcontaining 25 mg/ml ketamine, 2.5 mg/ml xylazine and 0.5 mg/ml acepromazine at 2.5 ml/g body weight. The EDL muscle wasgently dissected and mounted to an intact muscle test system(Aurora Scientific, Inc., Aurora, ON, Canada) (41). Briefly, theproximal tendon of the EDL muscle was secured to a dual-modeservomotor transducer and the distal tendon was attached to afixed post using a 4-0 suture (SofSilk USSC Sutures, Norwalk,CT, USA). Subsequently, the EDL muscle was submerged in a308C jacketed organ bath containing oxygenated (95% O2 and5% CO2) Ringer’s buffer. After 10 min equilibration, theoptimal length (Lo) of the EDL muscle was measured with anelectronic digital caliper (Fisher Scientific, Waltham, MA,USA). The maximum isometric tetanic force (Po) was measuredat 150 Hz. The muscle CSA was calculated according to the fol-lowing equation, CSA ¼ (muscle mass, in gram)/[(optimal fiberlength, in cm) × (muscle density, in g/cm3)]. A muscle densityof 1.06 g/cm3 was used in calculation. Specific muscle forcewas determined by dividing the maximum isometric tetanicforce with the muscle CSA. After tetanic force measurement,the muscle was rested for 10 min and then subjected to fiverounds of eccentric contraction injury according to our previouslypublished protocol (41). The percentage of force drop followingeach round of eccentric contraction was recorded. Data wererecorded and analyzed using the Lab View-based DMC andDMA programs (Version 3.12, Aurora Scientific, Inc.).

In situ examination of the TA muscle function

The TA muscle force was measured according to our publishedprotocol (12,41). Briefly, mice were anesthetized as describedabove. The TA muscle and the sciatic nerve were exposed.The mouse was transferred to a customer-designed thermo-controlled platform of the footplate apparatus (42). Subsequent-ly, twitch and tetanic forces were measured in situ with a305C-LR dual-mode servomotor transducer (Aurora Scientific,Inc.). Data recording and analysis were identical to methodsdescribed for the EDL muscle.

Evaluation of hindlimb muscle injury by chronictreadmill running

Mice were subject to chronic treadmill running as we describedbefore using an Exer 3/6 treadmill system (Columbus Instru-ments, Columbus, OH, USA) (18,41). Briefly, mice were accli-mated to the treadmill for 3 days and then challenged withtreadmill running twice a week for a total of 8 weeks. Each tread-mill session lasted 40 min including 10 min warm-up at thespeed of 8 m/min followed by 30 min running at a speed of12 m/min on a horizontal treadmill. At the end of 8-week tread-mill exercise, mice were euthanized and tetanic force of the EDLmuscle was measured as described above at the stimulation fre-quencies of 50, 80, 100 and 150 Hz. A separate set of mice (ageand sex-matched) that did not undergo exercise running was usedas controls for comparison. A statistically significant reductionof the EDL muscle specific force in exercised mice was consid-ered a positive indication for the presence of muscle damage dueto the loss of sarcolemmal nNOS (18).




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Evaluation of functional muscle ischemia duringhindlimb contraction

Femoral vascular conductance in mdx mice was evaluated using apulsed Doppler velocimeter (Indus Instruments, Webster, TX,USA) based on our previously published protocol (8,10).Briefly, mice were anesthetized with isoflurane (1.5–2.0% inoxygen) and instrumented with a carotid artery catheter tomeasure blood pressure, a right femoral artery catheter to delivernorepinephrine and a left femoral artery Doppler ultrasound flowprobe to measure hindlimb blood flow velocity. Core temperaturewas monitored using a rectal probe and was maintained at 378C.Femoral vascular conductance (mean blood flow velocity/meanblood pressure) responses to intra-arterial injection of thea-adrenergic vasoconstrictor norepinephrine (12.5–25 ng in avolume of 5–20 ml) were measured with the left hindlimb at restand during intermittent, tetanic contractions induced by sciaticnerve stimulation using 100 ms trains of pulses (100 Hz, 0.2 ms)at a rate of 30 trains per min (Model S88, Grass Instruments).Femoral vascular conductance responses to norepinephrine werecalculated by integrating the area under the response curve.

Statistical analysis

Data are presented as mean+ standard error of mean. Statisticalanalysis was performed with the SPSS software (IBM Corpor-ation, Armonk, NY, USA). For multiple group comparison,statistical significance was determined by one-way ANOVA fol-lowed by Bonferroni post hoc analysis. For two group compari-son, statistical significance was determined by the Student t-test.Difference was considered significant when P , 0.05.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

AUTHOR CONTRIBUTIONS

Conceived and designed experiments: D.D. and G.D.T.Performed experiments: Y.Z., Y.Y., L.L., C.H.H. andK.Z. Analyzed data: D.D., G.D.T. and Y.Z. Wrote the paper: D.D.

ACKNOWLEDGEMENTS

We thank Thomas McDonald for excellent technical assistance.We thank Yi Lai for helpful discussion.

Conflict of Interest statement. Y.Y. and D.D. hold a patent on thenNOS binding mini-dystrophin gene.

FUNDING

The study was supported by grants from the National Institutes ofHealth AR-49419 (D.D.), HL-91883 (D.D.) and AR-56221(G.D.T.) and Muscular Dystrophy Association (D.D.).

REFERENCES

1. Kunkel, L.M. (2005) 2004 William Allan award address. Cloning of theDMD gene. Am. J. Hum. Genet., 76, 205–214.

2. Yue, Y., Ghosh, A., Long, C., Bostick, B., Smith, B.F., Kornegay, J.N. andDuan, D. (2008) A single intravenous injection of adeno-associated virusserotype-9 leads to whole body skeletal muscle transduction in dogs. Mol.

Ther., 16, 1944–1952.

3. Bostick, B., Ghosh, A., Yue, Y., Long, C. and Duan, D. (2007) SystemicAAV-9 transduction in mice is influenced by animal age but not by the routeof administration. Gene Ther., 14, 1605–1609.

4. Lai, Y., Yue, Y. and Duan, D. (2010) Evidence for the failure ofadeno-associated virus serotype 5 to package a viral genome . or ¼8.2 kb.Mol. Ther., 18, 75–79.

5. England, S.B., Nicholson, L.V., Johnson, M.A., Forrest, S.M., Love, D.R.,Zubrzycka-Gaarn, E.E., Bulman, D.E., Harris, J.B. and Davies, K.E. (1990)Very mild muscular dystrophy associated with the deletion of 46% ofdystrophin. Nature, 343, 180–182.

6. Harper, S.Q., Hauser, M.A., DelloRusso, C., Duan, D., Crawford, R.W.,Phelps, S.F., Harper, H.A., Robinson, A.S., Engelhardt, J.F., Brooks, S.V.et al. (2002) Modular flexibility of dystrophin: implications for gene therapyof Duchenne muscular dystrophy. Nat. Med., 8, 253–261.

7. Brenman, J.E., Chao, D.S., Xia, H., Aldape, K. and Bredt, D.S. (1995) Nitricoxide synthase complexed with dystrophin and absent from skeletal musclesarcolemma in Duchenne muscular dystrophy. Cell, 82, 743–752.

8. Lai, Y., Thomas, G.D., Yue, Y., Yang, H.T., Li, D., Long, C., Judge, L.,Bostick, B., Chamberlain, J.S., Terjung, R.L. et al. (2009) Dystrophinscarrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma andenhance exercise performance in a mouse model of muscular dystrophy.J. Clin. Invest., 119, 624–635.

9. Thomas, G.D. and Victor, R.G. (1998) Nitric oxide mediatescontraction-induced attenuation of sympathetic vasoconstriction in ratskeletal muscle. J. Physiol., 506, 817–826.

10. Thomas, G.D., Sander, M., Lau, K.S., Huang, P.L., Stull, J.T. and Victor,R.G. (1998) Impaired metabolic modulation of alpha-adrenergicvasoconstriction in dystrophin-deficient skeletal muscle. Proc. Natl Acad.

Sci. USA, 95, 15090–15095.

11. Sander, M., Chavoshan, B., Harris, S.A., Iannaccone, S.T., Stull, J.T.,Thomas, G.D. and Victor, R.G. (2000) Functional muscle ischemia inneuronal nitric oxide synthase-deficient skeletal muscle of children withDuchenne muscular dystrophy. Proc. Natl Acad. Sci. USA, 97, 13818–13823.

12. Li, D., Yue, Y., Lai, Y., Hakim, C.H. and Duan, D. (2011) Nitrosative stresselicited by nNOSmu delocalization inhibits muscle force in dystrophin-nullmice. J. Pathol., 223, 88–98.

13. Lai, Y., Yue, Y., Liu, M., Ghosh, A., Engelhardt, J.F., Chamberlain, J.S. andDuan, D. (2005) Efficient in vivo gene expression by trans-splicingadeno-associated viral vectors. Nat. Biotechnol., 23, 1435–1439.

14. Judge, L.M., Haraguchi, M. and Chamberlain, J.S. (2006) Dissecting thesignalingand mechanical functions of the dystrophin-glycoprotein complex.J. Cell Sci., 119, 1537–1546.

15. Lai, Y., Zhao, J., Yue, Y. and Duan, D. (2013) alpha2 and alpha3 helices ofdystrophin R16 and R17 frame a microdomain in the alpha1 helix ofdystrophin R17 for neuronal NOS binding. Proc. Natl Acad. Sci. USA, 110,525–530.

16. Zhang, Y. and Duan, D. (2012) Novel mini-dystrophin gene dualadeno-associated virus vectors restore neuronal nitric oxide synthaseexpression at the sarcolemma. Hum. Gene Ther., 23, 98–103.

17. Odom, G.L., Gregorevic, P., Allen, J.M. and Chamberlain, J.S. (2011) Genetherapy of mdx mice with large truncated dystrophins generated byrecombination using rAAV6. Mol. Ther., 19, 36–45.

18. Li, D., Bareja, A., Judge, L., Yue, Y., Lai, Y., Fairclough, R., Davies, K.E.,Chamberlain, J.S. and Duan, D. (2010) Sarcolemmal nNOS anchoringreveals a qualitative difference between dystrophin and utrophin. J. Cell Sci.,123, 2008–2013.

19. Pastoret,C. and Sebille, A. (1995) Mdx mice show progressive weakness andmuscle deterioration with age. J. Neurol. Sci., 129, 97–105.

20. Lynch, G.S., Hinkle, R.T., Chamberlain, J.S., Brooks, S.V. and Faulkner,J.A. (2001) Force and power output of fast and slow skeletal muscles frommdx mice 6–28 months old. J. Physiol., 535, 591–600.

21. Duan, D. (2011) Duchenne muscular dystrophy gene therapy: lost intranslation? Res. Rep. Biol., 2, 31–42.

22. Hillier, B.J., Christopherson, K.S., Prehoda, K.E., Bredt, D.S. and Lim, W.A.(1999) Unexpected modes of PDZ domain scaffolding revealed by structureof nNOS-syntrophin complex. Science, 284, 812–815.




Dow

nloaded from



23. Wang, B., Li, J., Qiao, C., Chen, C., Hu, P., Zhu, X., Zhou, L., Bogan, J.,Kornegay, J. and Xiao, X. (2008) A canine minidystrophin is functional andtherapeutic in mdx mice. Gene Ther., 15, 1099–1106.

24. Yue, Y., Liu, M. and Duan, D. (2006) C-terminal truncated microdystrophinrecruits dystrobrevin and syntrophin to the dystrophin-associatedglycoprotein complex and reduces muscular dystrophy in symptomaticutrophin/dystrophin double knock-out mice. Mol. Ther., 14, 79–87.

25. Ghosh, A., Yue, Y. and Duan, D. (2011) Efficient transgene reconstitutionwith hybrid dual AAV vectors carrying the minimized bridging sequence.Hum. Gene Ther., 22, 77–83.

26. Ghosh, A., Yue, Y., Lai, Y. and Duan, D. (2008) A hybrid vector systemexpands aden-associated viral vector packaging capacity in a transgeneindependent manner. Mol. Ther., 16, 124–130.

27. Duan, D., Yue, Y. and Engelhardt, J.F. (2001) Expanding AAV packagingcapacity with trans-splicing or overlapping vectors: a quantitativecomparison. Mol. Ther., 4, 383–391.

28. Duan, D., Yue, Y., Yan, Z. and Engelhardt, J.F. (2000) A new dual-vectorapproach to enhance recombinant adeno-associated virus-mediated geneexpression through intermolecular cis activation. Nat. Med., 6, 595–598.

29. Lai, Y., Yue, Y., Bostick, B. and Duan, D. (2010) In Duan, D. (ed.), MuscleGene Therapy. Springer Science+Business Media, LLC, New York, pp.205–218.

30. Ghosh, A. and Duan, D. (2007) Expending adeno-associated viral vectorcapacity: a tale of two vectors. Biotechnology and Genetic EngineeringReviews, 24, 165–177.

31. Lostal, W., Bartoli, M., Bourg, N., Roudaut, C., Bentaib, A., Miyake, K.,Guerchet, N., Fougerousse, F., McNeil, P. and Richard, I. (2010) Efficientrecovery of dysferlin deficiency by dual adeno-associated vector-mediatedgene transfer. Hum. Mol. Genet., 19, 1897–1907.

32. Ghosh, A., Yue, Y., Shin, J.-H. and Duan, D. (2009) Systemic trans-splicingAAV delivery efficiently transduces the heart of adult mdx mouse, a modelfor Duchenne muscular dystrophy. Hum. Gene Ther., 20, 1319–1328.

33. Ghosh, A., Yue, Y., Long, C., Bostick, B. and Duan, D. (2007) Efficientwhole-body transduction with trans-splicing adeno-associated viral vectors.Mol. Ther., 15, 750–755.

34. Ghosh, A., Yue, Y. and Duan, D. (2006) Viral serotype and the transgenesequence influence overlapping adeno-associated viral (AAV)vector-mediated gene transfer in skeletal muscle. J. Gene Med., 8, 298–305.

35. Chamberlain, J.S. (2002) Gene therapy of muscular dystrophy. Hum. Mol.

Genet., 11, 2355–2362.

36. Chapman, V.M., Miller, D.R., Armstrong, D. and Caskey, C.T. (1989)Recovery of induced mutations for X chromosome-linked musculardystrophy in mice. Proc. Natl Acad. Sci. USA, 86, 1292–1296.

37. Sicinski, P., Geng, Y., Ryder-Cook, A.S., Barnard, E.A., Darlison, M.G. andBarnard, P.J. (1989) The molecular basis of muscular dystrophy in the mdxmouse: a point mutation. Science, 244, 1578–1580.

38. Shin, J.H., Yue, Y. and Duan, D. (2012) Recombinant adeno-associatedviral vector production and purification. Methods Mol. Biol., 798,267–284.

39. Zhong, L., Li, B., Mah, C.S., Govindasamy, L., Agbandje-McKenna, M.,Cooper, M., Herzog, R.W., Zolotukhin, I., Warrington, K.H. Jr., Weigel-VanAken, K.A. et al. (2008) Next generation of adeno-associated virus 2 vectors:point mutations in tyrosines lead to high-efficiency transduction at lowerdoses. Proc. Natl Acad. Sci. USA, 105, 7827–7832.

40. Yue, Y., Li, Z., Harper, S.Q., Davisson, R.L., Chamberlain, J.S. and Duan, D.(2003) Microdystrophin gene therapy of cardiomyopathy restoresdystrophin-glycoprotein complex and improves sarcolemma integrity in themdx mouse heart. Circulation, 108, 1626–1632.

41. Hakim, C.H., Li, D. and Duan, D. (2011) Monitoring murine skeletal musclefunction for muscle gene therapy. Methods Mol. Biol., 709, 75–89.

42. Hakim, C.H., Wasala, N.B. and Duan, D. (2013) Evaluation of musclefunction of the extensor digitorum longus muscle ex vivo and tibialis anteriomuscle in situ in mice. J. Vis. Exp., 73, e50183.




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Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy

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