458 external, non invasive monitoring of progressive cardiorespiratory dysfunction in a canine model of DMD

458 External, Non Invasive Monitoring of Progressive Cardiorespiratory Dysfunction in a Canine Model of DMD Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene[.]

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MUSCULO-SKELETAL GENE & CELL THERAPY II

polyclonally stimulated T-cells (p<0,05) in a 1:2 ratio Insertion of

MOG-reactive CAR into MSC made CNS-speci c MSCs that retained

their capacity to signi cantly suppress T-cell proliferation (p<0,001)

in a 1:2 ratio Further, when adding MOG+ cells to the co cultures

the engineered Tregs and MSCs (p<0,001) were still suppressive

Furthermore, engineered Tregs and MSCs were able to locate to

and transmigrate into the brain of mice 10 days post systemical i.p

injection In conclusion, genetically engineered suppressor cells

are able to localize to the CNS and to suppress the proliferation of

activated T-cells

Musculo-skeletal Gene & Cell Therapy II

454 First Generation AAVmicroutrophin Vector

Infused into the Isolated Pelvic Limb of a Canine

Model for Duchenne Muscular Dystrophy

Mihail Petrov,1 Marilyn Mitchell,1 Alock Malik,1 Andy Mead,1

Frederick Balzer,1 Leonard Su,1 Jacqueline Farag,1 Benjamin

Kozyak,1 Kapil Gopal,1 Charles Bridges,1 Janet Bogan,2 Martin

Childers,3 Joe Kornegay,2 Hansell Stedman.1

1 Surgery, University of Pennsylvania, Philadelphia, PA; 2 University

of North Carolina, Chapel Hill, NC; 3 Wake Forest Univ., Winston

Salem, NC.

Germline and somatic gene transfer of internally deleted dystrophin

and utrophin coding sequences into dystrophic mice has provided

evidence for phenotypic amelioration Myotrophic AAV vectors have

been shown to be amenable to a vascular route of administration,

suggesting that partial phenotypic correction should be feasible in

a large animal disease model To this end, we initially injected the

isolated pelvic limbs of dogs hemi- or homozygous for the GRMD

mutation (dystrophin intron 6 splice acceptor site) with a “ rst

generation” AAV6 vector containing a constitutive promoter/enhancer

driving transcription of a “microutrophin” cassette based on the wild

type cDNA sequence The afferent, transvenular extravasation route of

administration used recapitulates that previously shown to transduce

essentially 100% of the skeletal muscle  bers in the mature canine

leg (Su, Gopal, et al, 2005) A proportion of the dogs underwent

transient single agent immunosuppression using a protocol previously

shown to prevent inhibitory antibody formation in a canine model for

hemophilia B (Arruda, Stedman, et al, 2005) Dogs were injected with

vector at one of three doses Follow up studies for force transduction

were performed by a group of investigators blinded as to the AAV

dose, identity of the injected limb, and the presence or absence of

prior immunosuppression In a group of dogs receiving the highest

dose of AAV.utrophin-1 (10E13.5vg/kg), the ratio of torque developed

by the treated vs untreated limb is 1.07+/-.2 Five of six dogs in this

group showed  exion strength improvement on the treated side If

one disallows the data from the one confounding dog on the grounds

that a minor technical problem occurred (electrode migration during

force transduction, as suspected from the discrepancy between serial

measurements in this dog) the average ratio among remaining dogs

is 1.15+/-.08 Importantly, with this combination of treatment and

immunosuppressive regimen the muscles did not become weaker, as

one might have expected with an AAV-induced myositis Interestingly,

data on other non-immunosuppressed groups of dogs suggest that the

vector might have caused a subclinical myositis Among dystrophic

dogs receiving even lower doses of AAV.utrophin-1 without

cyclophosphamide, the ratio of treated to untreated limb torque

was 0.94+/-0.05 Moreover, among non-dystrophic dogs receiving

the unrelated AAV.F.IX (containing a “self” transgene) without

immunosuppression, the ratio of treated to untreated limb strength

was 0.85+/-0.06 In conjunction with emerging trends in AAV-based

clinical investigation, these observations heighten the impetus to

formally address the prevention of cellular immune response directed

against input AAV capsid antigens

455 Characterization of A20 as an Inhibitor of NF-țB Activation in Dystrophic Mice

Rakshita Charan,1 Paula R Clemens.1,2

1 Department of Neurology, University of Pittsburgh, Pittsburgh, PA; 2 Department of Veterans Affairs Medical Center, Veterans

Affairs, Pittsburgh, PA.

Of all muscular dystrophies, Duchenne muscular dystrophy (DMD)

is the most common affecting about 1 in 3500 male births worldwide

The disease is caused by mutations in the gene dystrophin, the protein product of which is required for muscle structure and stability Studies suggest that the lack of structural support in dystrophin-de cient muscle  bers may be responsible for muscle pathology in progressive muscular dystrophy It is also known that nuclear factor-kappa B (NF-κB), which is a nuclear transcription factor, is up regulated in dystrophic muscle in DMD patients as well as in the mouse model for DMD (mdx) NF-κB regulates several genes responsible for stress responses, cell survival and various in ammatory conditions

Thus, the up regulation of this transcription factor is thought to activate protein degradation and cause chronic in ammation in skeletal muscle Furthermore, NF-κB downregulates myogenic regulatory factors and this process likely interferes with muscle regeneration Attenuating NF-κB activation in these dystrophic mice has been shown to improve muscle stability and strength Strategies

of inhibition of NF-κB activation are being actively pursued as a therapeutic option for DMD One of the NF-κB pathway attenuators, A20 is a deubiquitinating enzyme, known to inhibit NF-κB activation

by deubiquitinating RIP1; ubiquitination of RIP1 is essential for NF-κB activation

Our aim is to characterize A20 in skeletal muscle and establish its role as a potential therapeutic target as attenuator of the NF-κB pathway activation in DMD We show that blocking of A20 using A20siRNA increases NF-κB activation in mdx as well as control C57BL/10 mice myotubes We further characterized localization of A20 in muscle and established that A20 is expressed predominantly

in fast-twitch muscle  bers Interestingly, we also observed that in mdx muscle, A20 is over-expressed in regenerating  bers To study the localization of A20 through the life of the mdx mouse, we did

a time-pro le assessment of A20 expression and compared it with control mice We see an increase in A20 protein expression during the 7-10 week time period in mice, which is correlative of the ages when severe degeneration and regeneration cycles take place in mdx mice This is the  rst observation of a correlation between expression

of an NF-κB inhibitor and pathology of DMD

Our studies support the utility of therapeutic manipulation of A20 to promote NF-κB inactivation in dystrophic muscle  bers and provide

a potential therapy for DMD

456 Plasticity of Skeletal Muscle Cells during Muscle Injury and Repair – A Dedifferentiation

Study

Xiaodong Mu,1,2 Hairong Peng,2 Johnny Huard,2,3 Yong Li.1,2,3,4

Center (S.C.R.C.),, Children’s Hospital of UPMC, Pittsburgh, PA;

2 Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 3 Department of Bioengineering, University of

Pittsburgh, Pittsburgh, PA; 4 Department of Pathology, University

of Pittsburgh, Pittsburgh, PA.

INTRODUCTION: In the skeletal muscle system of certain

amphibians, dedifferentiation occurs after muscle injury and mononuclear cells are released from myo bers These mononuclear cells can then serve as an source of precursor cells for effective muscle regeneration However, whether a similar dedifferentiation process occurs in mammals remains largely a mystery, and it was generally believed that the main sources of muscle precursors for ef cient

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MUSCULO-SKELETAL GENE & CELL THERAPY II

muscle regeneration are muscle stem cells and satellite cells Here in this study, transgenic myoblasts (with the Cre/Lox-β-galactosidase system) were transplanted into skeletal muscle of adult mouse model for determining the process of dedifferentiation after injury

METHODS: Cre-Lox system: Myocytes from a normal mouse were

isolated and transfected with a Cre-expressing retrovirus to generate M-Cre The M-Cre were co-cultured with another population of myocytes isolated from 129-Gt(ROSA)26Sortm1Sho (Lox-βgeo) reporter mice (M-Lox) Fusion of M-Cre and M-Lox resulted in Cre recombinase-mediated cleavage of Lox sites and induction of β-gal

expression in resulting fused myotubes Muscle injury: 3 weeks after

transplantation of Cre-Lox cells into gastrocnemius (GM) muscle

of SCID mice (C57BL/6J), lacerations were performed GM of one

leg, and the GM muscle of the other leg serves as control Isolation

of muscle cells: 4 days after muscle injury, the cells were isolated

with pre-plate technique The isolated cells were cultured in the proliferation medium [DMEM supplemented with 10% Fetal Bovine Serum (FBS), 10% Horse Serum (HS), 1% Penicillin-Streptomycin

antibiotics, and 0.5% chicken essential extract (CEE)] RESULTS:

1 The function of Cre-Lox system in differentiated myotubes

in vitro: 4 days after myogenic differentiation, β-gal expression was exclusively observed in multinuclear myotubes in the mixed culture of Cre-cells and Lox cells 2 Cre/Lox-β-galactosidase cell transplantation and follow up muscle injury resulted in β-gal positive mononucleated cells, indicating the occurring of muscle cell dedifferentiation 3 β-galactosidase positive cells from injured muscle stay in different populations (myoblasts, satellite cells, and

muscle stem cells) DISCUSSION: Our results demonstrate that

dedifferentiation may occur in injured skeletal muscle The process

of dedifferentiation in the differentiated muscle cells is proved by our Cre-Lox system Mononuclear cells released from differentiated (β-galactosidase positive) myocytes, which is a single population of cells at beginning, were shown to have developed into different cell populations including MyoD+ myoblasts, Pax-7+ satellite cells, and Sca-1+/CD34+ muscle stem cells Therefore, the dedifferentiation process may contribute to muscle regeneration during muscle injury

and repair Acknowledgement: Authors acknowledge funding from

NIH and DOD

457 Increased Muscle Regeneration in

Hindlimb and Diaphragm of mdx Mice Treated

with AAV9 Mini-Dystrophin and Octalysine-NEMO Binding Domain Peptide

Daniel P Reay,1 Geno Raggi,1 Bing Wang,2 Xiao Xiao,5 Paul D

Robbins,3 Paula R Clemens.1,4

1 Neurology, University of Pittsburgh, Pittsburgh, PA; 2 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 3 Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA; 4 Department of Veteran’s Affairs, Pittsburgh, PA; 5 School of Pharmacy, University of North Carolina, Chapel Hill, NC.

The loss of a functional, membrane-localized dystrophin protein

is the primary cause of Duchenne muscular dystrophy (DMD) The muscle in ammatory in ltrates and failure of muscle regeneration that are secondary to dystrophin loss have recently been shown to be due,

in part, to activation of the nuclear factor κB (NF-κB) Dystrophic muscle shows substantially increased nuclear accumulation of

NF-κB, which is well known to regulate cytokines, other in ammatory molecules and myogenic proteins Toward the development of therapy for DMD, we designed an experiment to test the combination of dystrophin gene replacement and inhibition of NF-κB activation

For dystrophin gene replacement therapy, we utilized an AAV9 vector to systemically deliver a mini-dystrophin transgene to 3

day old neonatal dystrophin-de cient mdx mice Beginning at 4

weeks of age, mice were then treated with tri-weekly injections of octalysine-NEMO binding domain peptide (8K-NBD), to inhibit

NF-κB activation Mice treated with either AAV9 mini-dystrophin vector alone or AAV9 mini-dystrophin vector plus 8K-NBD peptide exhibited mini-dystrophin expression and reduced necrosis in the quadriceps and diaphragm, but mice treated with the AAV9 mini-dystrophin vector plus 8K-NBD peptide additionally demonstrated increased levels of muscle regeneration in quadriceps and diaphragm EMSA analysis for NF-κB con rmed decreased levels of nuclear

NF-κB in 8K-NBD-treated mdx mice Our data suggest that high

levels of mini-dystrophin gene transfer are required to demonstrate

a reduction in the nuclear accumulation of NF-κB in muscle of mdx mice treated with vector alone Overall, treatment of mdx mice with

AAV9 mini-dystrophin gene replacement therapy in combination with NBD peptide/NF-κB inhibitory therapy may provide critical insight into potential treatments for DMD

458 External, Non-Invasive Monitoring of Progressive Cardiorespiratory Dysfunction in a Canine Model of DMD

Andrew Mead,1 Alock Malik,1 Mihail Petrov,1 Martin Childers,2

Janet Bogan,3 Joseph Kornegay,3 Hansell Stedman.1

1 University of Pennsylvania, Philadelphia, PA; 2 Wake Forest University, Winston-Salem, NC; 3 University of North Carolina, Chapel Hill, NC.

The GRMD (Golden Retriever Muscular Dystrophy) model for Duchenne Muscular Dystrophy (DMD) mimics human disease progression with regard to both histopathology and locomotive function However, unlike DMD, the GRMD model exhibits a high degree of variability in both the severity and speed of progression, even among littermates This aspect of the model poses challenges for assessing efficacy in pre-clinical therapeutic trials of gene therapy, and requires further improvement in quantitative measures

of disease progression Electrical stimulation of distal limb muscles has provided the most reliable quantitative data, however this requires serial episodes of general anesthesia, with attendant risks of cardiopulmonary complication in the dystrophic dogs Moreover, the data are limited to locomotive muscle function Most non-invasive means of assessment, such as running times, are unreliable due to their volitional nature, and fail to dissect the cardiac, respiratory and locomotive components of disease progression Here we investigate the use of a non-invasive device that records EKG, 3D accelerometry, and 2-band plethysmography in a wireless package that has negligible effect on animal behavior, the “Lifeshirt” (Vivometrics) In particular we simultaneously examine cardiac and respiratory function, two physiological parameters relevant to longevity in DMD 1 We hypothesize that the progressive loss of ventilatory reserve caused by degeneration of the diaphragm will manifest, in the GRMD model, as a progressive respiratory paradox, (i.e asynchrony in the chest wall and abdominal wall excursion) during and immediately after mild exertion The degree of paradox will correlate with trans-diaphragmatic pressure development during stimulated spontaneous breathing and phrenic nerve stimulation at the time of necropsy 2 Progressive heart failure, also a hallmark of DMD in humans, will manifest  rst as parasympathetic withdrawal measured by depressed respiratory sinus arrhythmia in affected dogs

as compared to unaffected littermates Decreased RSA would be expected to correlate with impaired cardiac contractility as measured

by more direct means, e.g Langendorff isolated perfused heart studies performed post mortem

Data obtained using the “Lifeshirt”system on GRMD pups reveals that by 6 months of age there are early indicators of respiratory and cardiac dysfunction In preliminary studies of 11 dystrophic and 7 normal dogs under 8 months of age RSA was signi cantly reduced in the affected group (p<.006), and respiratory phase angle was higher (p<.05) Affected pups were also marked by the complete absence

of diaphragm-intensive panting behavior These differences between

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MUSCULO-SKELETAL GENE & CELL THERAPY II

normal and affected animals, and among affected animals at different

stages of disease progression increased markedly after moderate

voluntary exercise Standardization of these approaches will enhance

the GRMD model’s role in translational studies of gene therapy for

cardiac and respiratory muscle disease in DMD

459 The Polyproline Site in Hinge 2 In uences

the Functional Capacity of Truncated Dystrophins

Glen B Banks, Luke M Judge, James M Allen, Jeffrey S

Chamberlain

Department of Neurology, University of Washington, Seattle, WA.

Mutations in dystrophin can lead to Duchenne muscular dystrophy

or the more mild form of the disease, Becker muscular dystrophy

(BMD) The hinge 3 region in the rod domain of dystrophin is

particularly prone to deletion mutations In-frame deletions of hinge

3 are predicted to lead to BMD, however the severity of disease can

vary considerably Here we performed extensive structure-function

analyses of truncated dystrophins with modi ed hinges and

spectrin-like repeats in mdx mice We found that the polyproline site in hinge 2

profoundly in uences the functional capacity of a microdystrophin

∆R4-R23/∆CT with a large deletion in the hinge 3 region Inclusion of

polyproline in microdystrophin∆R4-R23/∆CT led to small myo bers (12%

smaller than wild-type), Achilles myotendinous disruption, ringed

 bers and aberrant neuromuscular junctions in the mdx gastrocnemius

muscles Replacing hinge 2 of microdystrophin∆R4-R23/∆CT with hinge

3 signi cantly improved the functional capacity to prevent muscle

degeneration, increase muscle  ber area and maintain the junctions

We conclude that the rigid α-helical structure of the polyproline site

signi cantly impairs the functional capacity of truncated dystrophins

to maintain appropriate connections between the cytoskeleton and

extracellular matrix

460 Combining Gene and Stem Cell Therapy in

the Treatment of Dysferlinophaties

Daniele Parolini,1 Claire Navarro,1,2 Andrea Farini,1 Mirella

Meregalli,1 Marzia Belicchi,1 Paola Razini,1 M Krahn,2 Louis

Garcia,3 Nicolas Lévis,2 Yvan Torrente.1

1 Dept of Neurological Sciences, Università degli Studi di Milano,

Milan, Italy; 2 Laboratoire de Genetique Moleculaire, Hopital

d’Enfants de la Timone, INSERM U910 Genetique Medicale

INSERM/UPMC, Institut de Myologie, Faculté de Médecine Pierre

et Marie Curie, Paris, France.

Mutations in gene encoding dysferlin are involved in two main

muscular dystrophies: Miyoshi myopathy and Limb-Girdle Muscular

Dystrophy 2B Both diseases are characterized by progressive

weakness and skeletal muscle wasting Dysferlin is expressed in

skeletal and cardiac muscles, where its main function is membrane

repair So far, no treatment is available and development of effective

therapies remains a big challenge New hopes are coming from stem

cell and gene therapies Based on work developed in DMD, we

investigated the feasibility of stem cell engineering by exon-skipping

in dysferlinopathies We focused our efforts on a patient carrying a

deletion in exon 22 found at heterozygous composite state with a large

deletion (∆25-29) predicted to be in-frame According to the absence

of protein observed, we supposed a destabilization and/or degradation

of this predicted in-frame truncated mRNA and/or protein In this

study both exons 22 and 23 needed to be removed to restore the

ORF and allow production of a truncated functional protein As

proof of principle of the exon-skipping feasibility, we  rstly tested

if the skipped dysferlin product can be correctly expressed To

achieve this aim, constructs encoding deleted forms of dysferlin were

transfected into HEK and 3T3 cells The truncated dysferlin ∆22-23,

mimicking the skipped allele of our patient, was detected in WB,

correctly addressed to the membrane and functional Interestingly, also the protein ∆25-29 was produced, suggesting that the absence

of dysferlin in our patient is most probably due to mRNA instability

Finally, a third deleted form was produced carrying the deletion

∆22-29, suggesting that all these exons can be removed without affect dysferlin expression We then designed antisense oligonucleotides (AONs) able to target acceptor or donor splice sites as well as ESE sequences of exons 22 and 23 Their ability to skip exons of interest was tested on human normal myoblasts and circulating CD133 stem cells isolated from the patient Unfortunately, very low skipping efficiency was observed and results were not reproducible To overcome exon skipping dif culties, we developed a strategy based

on complete dysferlin delivery by lentivirus vector in blood-derived

CD133 stem cells isolated from the same and unskippable patients

The produced vector allowed dysferlin expression both in vitro, in

human  broblasts and mononucleated cells from our patients, and

in vivo after intramuscular injection of transduced CD133 stem

cells in the scid/blAJ mouse model We strongly believe that the

combination of gene and stem cell therapy represent a useful tool for new therapeutic approaches in dysferlinopathies Moreover, lentivirus

carrying complete dysferlin can be useful to bypass all unskippable

mutations, as those located in the transmembrane domain or other essential parts of the gene

461 Deletion of Functional Domains of Dp116 Leads to Altered Sarcolemmal Expression and Subcellular Localization When Expressed in Skeletal Muscle

Andrea L Arnett,1 Guy L Odom,1 Kristy Boyle,1 Eric Finn,1

Jeffrey S Chamberlain.1

1 Neurology, University of Washington, Seattle, WA.

Mice deficient in both dystrophin and utrophin (mdx:utrn -/-) exhibit a phenotype similar to that seen in DMD patients, including severe muscle wasting, skeletal deformities, joint contractures, and premature death In these mice, the absence of both dystrophin and utrophin leads to extremely low expression of members of the dystrophin-glycoprotein complex (DGC) We have discovered that

expression of the Dp116 isoform of dystrophin on the mdx:utrn

-/-background leads to a dramatic increase in lifespan and muscle mass

In contrast, Dp116 expression has minimal effect on mdx mice Dp116

is a non-muscle isoform of dystrophin that lacks the actin-binding domains and the majority of the rod domain found in the full-length dystrophin isoform Thus, it is postulated that Dp116 has no direct mechanical link to the actin cytoskeleton, but can still assemble and stabilize the DGC at the sarcolemma To decipher the mechanism through which Dp116 is contributing to phenotypic improvement in

mdx:utrn -/- mice, we have generated a series of staggered deletions

in the Dp116 cDNA utilizing recombinant PCR These constructs have been inserted into recombinant adeno-associated viral (rAAV6)

vectors and injected into mdx and mdx:utrn -/- mice for evaluation in vivo We have observed unique patterns of intracellular localization that correlate with the deletion of speci c functional domains of Dp116 In particular, the intracellular localization of Dp116 is altered

in the absence of either the WW or ZZ domains, which facilitate interaction with β-dystroglycan Deletion of the ZZ domain (∆ZZ) abolishes sarcolemmal association and results in diffuse, cytoplasmic distribution of Dp116 The ∆ZZ construct did not restore expression of other components of the DGC to the sarcolemma The WW deletion (∆WW) mutant retains a strong association with the sarcolemma, but simultaneously accumulates in cytoplasmic aggregates with long-term expression in mdx muscle In contrast to the ∆ZZ construct, expression

of the ∆WW mutant can restore sarcolemmal expression of the DGC

Other deletions also result in unusual localization patterns unique

to speci c domains These results suggest that multiple domains of

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MUSCULO-SKELETAL GENE & CELL THERAPY II

Dp116 are necessary for proper localization and function of dystrophin and the DGC, and have implications for the design of truncated dystrophins useful for cell and gene therapies of DMD

462 A New Therapeutic Approach for Duchenne Muscular Dystrophy: Restoration of the Dystrophin Reading Frame with a Meganuclease

Jacques P Tremblay,1 Pierre Chapdelaine,1 Christophe Pichavant,1

Joel Rousseau,1 Frédéric Pâques.2

2 Cellectis Genome Surgery, Cellectis S.A., Romainville, France.

Mutations in Duchenne muscular dystrophy are either inducing a nonsense codon or a frame shift Meganucleases can be engineered

to induce double strand break at speci c DNA sequences These breaks are repaired by Non Homologous End Joining (NHEJ), which results in insertions or deletions (Indels) of a few base pairs To verify whether meganucleases could be used to restore the normal reading frame of a dystrophin gene with a frame shift mutation, we have inserted a 29 bp sequence containing a target sequence for a meganuclease The co-transfection in 293FT cells of the target micro-dystrophin plasmid and of the appropriate meganuclease restored the dystrophin expression Meganucleases also restored micro-dystrophin expression in myoblasts and in muscle  bers in vivo The mutation of the targeted micro-dystrophin sequence was con rmed by PCR ampli cation followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons These experiments are thus a proof of principle that meganucleases engineered to target appropriate sequences in dystrophin gene would be able to restore the normal reading frame of that gene in DMD patients with out of frame deletion Meganucleases targeting a sequence including or near nonsense mutation could also be used to delete it

463 Regulatory Cassettes Derived from the Slow Troponin I Gene Confer High-Level Expression in Skeletal Muscle after Gene Transfer Mediated by Helper-Dependent Adenovirus and Recombinant AAV

Rénald Gilbert,1,2 Mehdi Bendjelloul,1,2 Yué Zeng,1,2 Claude Guérin,2 Nancy Larochelle,2 George Karpati,2 Bernard Massie,1,3

Josephine Nalbantoglu.2

1 Genomics & Gene Therapy Vectors, Biotechnology Research

Group, Montreal Neurological Institute, Montreal, QC, Canada;

Montréal, Montreal, QC, Canada.

Viral vectors used for gene replacement therapy of muscle diseases, such as Duchenne muscular dystrophy, require regulatory elements that can confer strong and muscle-speci c expression of the therapeutic gene product To achieve this goal, we generated regulatory cassettes by linking three (∆USEx3) or four (∆USEx4) copies of the truncated 60-pb upstream enhancer (∆USE) of human slow troponin I gene ∆USE has been reported to drive pan-muscle speci c expression in transgenic mice In addition, we have previously shown, using naked DNA, that ∆USEx3 confers strong and skeletal

muscle-speci c expression in cell culture and in vivo To evaluate

the activity of ∆USEx3 and ∆USEx4 in the context of viral vectors,

we constructed helper-dependent adenovirus (HD) expressing β-galactosidase (β-gal) regulated by ∆USEx3, ∆USEx4, or by the hybrid CMV enhancer/β-actin (CB) promoter We also constructed recombinant AAV vectors expressing the green  uorescent protein (GFP) regulated by ∆USEx4 or CMV The β-gal activity of

HD-∆USEx3(β) and HD-∆USEx4(β) was 1% of HD-CB(β) in cultured non-muscle cells and 20 to 90% in differentiated myotubes After

intramuscular injection of normal and mdx mouse muscle, the number

of transduced  bers and the β-gal activity of HD-∆USEx3(β) and HD-∆USEx4(β) corresponded to 20 to 80% of the value obtained with HD-CB(β) Notably, the number of transduced  bres after

intramuscular injection of AAV-∆USEx4(GFP) in adult mdx muscle

was similar to the value obtained under the same conditions using AAV-CMV(GFP) In summary, the strength, muscle speci city and small size of ∆USEx3 and ∆USEx4 render them very attractive for gene transfer applications in skeletal muscle

464 Cell Therapy of Muscular Dystrophy with Engineered Cd133+ Cells

Andrea Farini,1 Mirella Meregalli,1 Daniele Parolini,1 Marzia Belicchi,1 Simona Maciotta,1 Paola Razini,1 Joao da Silva Bizario,2

Luis Garcia,3 Nereo Bresolin,1 Yvan Torrente.1

of Milan, Università degli Studi, Dino Ferrari Center, Milan,

Pierre et Marie Curie, UMR S 787, INSERM/UPMC, Institut de Myologie, Paris, France.

DMD is a genetic disease caused by mutations in dystrophin gene Forced exclusion (skipping) of a single or multiple exons can restore the reading frame, giving rise to a shorter, but functional dystrophin protein We selected the GRMD dog, that shares with DMD patient progressive clinical signs and severe myopathy with contractures and premature death We isolated CD133+ cells from skeletal muscle biopsies of GRMD dogs and we transduced them with lentiviral vectors constructed to convey antisense oligonucleotides able to eliminate the mRNA segment from exon 6 to 8 Under appropriate sedation, the dogs received arterial systemic injections through a catheter introduced in the left femoralis artery and reached the aortic arch at the level of the left subclavia: cells were released mainly in the two large arteries under  uoroscopic guidance in order to provide the whole body musculature Serial injections of the engineered CD133+ cells do not stimulate an immunoreaction in the treated dogs The delivery of these cells results in a partial recovery of stiffness and ambulation disability of the treated dogs The muscle biopsies of the transplanted dogs showed clusters of dystrophin positives  bers This approach should offer a preclinical evidence for future therapies based on autologous transplantation

465 Improved Regenerative Capacity of Adult Stem Cells Isolated from Injured Skeletal Muscle of Mouse

Xiaodong Mu, Ian Bellayr, Haiying Pan, Yong Li

The Laboratory of Molecular Pathology, Stem Cell Research Center (S.C.R.C.), Children’s Hospital of UPMC, University

of Pittsburgh, Pittsburgh, PA; Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Department

of Bioengineering, Department of Pathology, Pittsburgh, PA; Department of Pathology, Pittsburgh, PA.

INTRODUCTION: Isolation of muscle derived stem cells

(MDSCs) has been popularly conducted with pre-plate technique according to their slow adhering characteristics on collagen-coated surface, and the transplantation of MDSCs has been veri ed to be greatly ef cient for stem-cell based therapies However, most of previous studies on muscle stem cells have been focused on the cells from normal muscle, in which the population of MDSCs is in fact very limited, and the isolation of MDSCs from injured muscle has not been described yet Injuries to the muscle can result in the activation, proliferation, and even profound phenotypic modi cation

of multiple cell types, including MDSCs In this study, with pre-plate method, MDSCs were isolated from injured muscle, and some of their characteristics have been compared with MDSCs from normal

muscle METHODS: Isolation of slow adhering cells: MDSCs were