249 Long Term Transgene Expression from High Capacity Adenoviral Vectors Delivered to Fetal Muscle In Utero Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ����������� �!���[.]
GENE THERAPY FOR THE NERVOUS SYSTEM I oligonucleotides were the chimeraplast (MDX7¹) and the DNA oligonucleotide that was designed to anneal with the coding strand (MDX7³) The chimeric and DNA oligonucleotides also corrected the mdx5cv mutation in vivo as determined by the restoration of dystrophin expression The expression of dystrophin was assessed as early as weeks after injection and was stable for at least months after injection Our studies provide evidence that oligonucleotide-mediated gene correction is a feasible approach to the treatment of certain genetic disorders in which long-term gene expression is required Thus this technology has the potential to be a viable, non-viral approach to stable restoration of gene expression GENE THERAPY OF MUSCLE DISEASES 249 Long-Term Transgene Expression from High-Capacity Adenoviral Vectors Delivered to Fetal Muscle In Utero 248 Correction of the Dystrophin Gene Mutation in the mdx5cv Mouse Model of Duchenne Muscular Dystrophy Mediated by Chimeric and DNA Oligonucleotides In Vitro and In Vivo Roberto Bilbao,1 Daniel Reay,1 Volker Biermann,2 Christoph Volpers,2 Zhilong Jiang,1 Stefan Kochanek,2 Paula R Clemens.1,3 Neurology, University of Pittsburgh, Pittsburgh, PA; 2Center for Molecular Medicine, University of Cologne, Cologne, Germany; Department of Veterans Affairs Medical Center, Pittsburgh, PA Carmen Bertoni,1 Thomas A Rando.1 Department of Neurology, Stanford University Medical Center, Stanford, CA, United States Gene correction represents an appealing option for the treatment of genetic disorders due to the prospect of permanent restoration of gene expression We have investigated the possibility of inducing single base pair alterations at the genomic level to restore the expression of dystrophin in mouse models of Duchenne muscular dystrophy, a severe muscle disease caused by mutations in the dystrophin gene We have shown the ability of chimeric RNA/DNA oligonucleotides (chimeraplasts) to correct a point mutation in the dystrophin gene in the mdx mouse We have also shown the feasibility of using chimeraplasts to mutate a base in an intron/exon boundary of the dystrophin gene to alter splicing, an approach that may be applicable to a variety of dystrophin gene defects We have now compared the correction ability of chimeraplasts to that of DNA oligonucleotides in muscle cells of the mdx5cv mouse This model has a point mutation in exon 10 of the dystrophin gene that creates a cryptic splice site Exon 10 is thus aberrantly spliced resulting in alteration of the dystrophin coding sequence leading to a lack of dystrophin expression We have designed a targeting chimeraplast (MDX7¹) and targeting DNA oligonucleotides (MDX7² and MDX7³) to specifically correct the mdx5cv mutation Each oligonucleotide is perfectly homologous to the region of exon 10 of the mdx5cv dystrophin gene containing the mutation, except for a mismatch at the mutated base The chimeric oligonucleotide is designed to pair with both strands of the DNA; the DNA oligonucleotides are designed to pair with either the coding (MDX7³) or the non-coding strand (MDX7²) As controls, we have used a chimeraplast (MDX8¹) and DNA oligonucleotides (MDX8² and MDX8³) identical to the targeting oligonucleotides but lacking the mismatch with the mdx5cv mutation Fluorescently labeled oligonucleotides are efficiently taken up in muscle precursor cells in vitro using all types of oligonucleotides Fluorescence persists longer in cells transfected with MDX7² or MDX7³, suggesting that DNA oligonucleotides have increased stability compared to chimeraplasts Restoration of dystrophin expression was assessed at the mRNA and protein level All targeting oligonucleotides were capable of restoring dystrophin expression, while control oligonucleotides had no effect Gene correction was demonstrated at the genomic level in cells transfected with targeting oligonucleotides Quantitative RT-PCR indicated that the level of gene correction varied between 0.2 to 5% The most efficient S98 In utero gene delivery holds promise for the treatment of hereditary diseases such as Duchenne muscular dystrophy (DMD) To date, efficient transduction has been achieved using firstgeneration adenoviral vectors Due to the large size of the dystrophin gene cDNA (14 kD), gene transfer of the full-length cDNA will require a vector with a larger insert capacity such as the high-capacity adenoviral (HC-Ad) vector In this study, we analyzed the longevity of transgene expression achieved by direct HC-Ad vector-mediated gene delivery to muscle in utero We also studied the efficiency of muscle gene delivery by HC-Ad vectors with intravascular delivery We first evaluated the transduction levels in muscle after intramuscular delivery of an HC-Ad vector carrying the lacZ gene (AdGS46) to fetal C57BL/6 mice 16 days after conception (E-16) Hind limb muscles were collected and months after infection and analyzed for β-galactosidase (β-Gal) expression by the onitrophenyl-β-D-galactopyranoside (ONPG) assay High levels of transgene expression in muscle were found We also investigated intravascular delivery of HC-Ad vector to C57BL/6 E-16 fetuses and observed high transduction efficiency in limb muscles In addition, higher survival rates were observed in those mice transduced with an HC-Ad vector as compared to a first-generation Ad vector To assess the potential of HC-Ad vector-mediated gene transfer to fetal muscle in a therapeutic model, we performed intramuscular injections of an HC-Ad vector carrying the dystrophin gene (AdDYS) to E-16 mdx mice, the animal model for DMD Immunohistochemical staining showed dystrophin expression in muscle of mdx mice transduced in utero with AdDYS Our results demonstrate that 1) Long-term transgene expression can be achieved by HC-Ad vector-mediated gene delivery to fetal muscle; 2) The HC-Ad vector can deliver full-length dystrophin to fetal muscle in utero Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy GENE THERAPY OF MUSCLE DISEASES 250 A Novel Approach To Identify Patients with Duchenne Muscular Dystrophy Caused by Stop Codon Mutations Using Aminoglycoside Antibiotics Shigemi Kimura,1 Tishihiko Miyagi,1 Takashi Hiranuma,1 Kowashi Yoshioka,1 Shiro Ozasa,1 Kaori Ito,1 Makoto Matsukura,1 Makoto Ikezawa,1 Masafumi Matsuo,2 Yasuhiro Takeshima,3 Teruhisa Miike.1 Department of Child Development, Kumamoto University School of Medicine, Kumamoto, Kumamoto, Japan; 2Division of Molecular Medicine, Kobe University Graduate School of Kobe, Kobe, Hyogo, Japan; 3Department of Pediatrics, Kobe University Graduate School of Kobe, Kobe, Hyogo, Japan Intro: Aminoglycoside antibiotics have been found to suppress premature stop codons located in the defective dystophin gene in mdx mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD) However, it is very difficult to find patients that are applicable for this therapy, because: 1) only to 10% of DMD patients have nonsense mutations in the dystrophin gene, 2) it is challenging to find nonsense mutations in the gene because dystrophin cDNA is very long (14kb), and 3) the efficiency of aminoglycoside-induced read-through is dependent on the type of nonsense mutation Recently, our research has focused on MyoD, a transcriptional factor that has the ability to differentiate fibroblasts into myotubes in vitro Adenoviral vectors encoding MyoD, regulated by CAG Promoter (AdMyoD), can efficiently transduce fibroblasts to express MyoD In this study, we introduce an easy system to identify patients for this therapy and report for the first time, that dystrophin expression was detected in myotubes of DMD patients using gentamicin Methods:Fibroblasts were isolated from six DMD patients In patient 1, a deletion of exons 48~50 in the dystrophin gene resulted in an out of frame pattern of the gene Patients 2-6 had nonsense mutations in the dystrophin gene; the stop codon is TGA for patients 2-4 and TAA for patients and Control fibroblasts were isolated from a non-DMD patient The fibroblasts were infected in vitro with AdMyoD using a multiplicity of infection (MOI) of 100 Following infection, the cells were cultured in DMEM supplemented with 2% FBS and 300 mg/ml of gentamicin At weeks postinfection, the dystrophin expression was analyzed by dystrophin staining and Western blotting analysis Results:The in vitro immunofluorescence staining and Western blot analysis for dystrophin showed that dystrophin expression was not detected in the myotubes of patient (deletion of dystrophin gene) cultured with and without gentamicin In contrast, dystrophin expression was detected in myotubes of patients 2, and (stop codon mutation TGA) cultured with gentamicin, but not detected in myotubes cultured without genatamicin Interestingly, dystrophin expression was not observed in myotubes from patients and with the stop codon mutation TAA, in spite of being cultured with gentamicin Dystrophin expression was detected in control myotubes from fibroblasts of a non-DMD patient after culturing them with and without gentamicin Discussion:We have developed a system to identify DMD patients caused by stop codon mutations in the dystrophin gene that are eligible for gentamicin treatment By monitoring dystrophin expression of myotubes differentiated from fibroblasts infected with AdMyoD and cultured in gentamicin, we are able to determine which patients will benefit from such treatments In addition, our results show that this system for the aminoglycoside treatment is far more effective for DMD patients that have nonsense mutation TGA than for patients that have nonsense mutation TAA Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy 251 Prolonged Dystrophin Expression and Functional Correction of mdx Mouse Muscle Following Gene Transfer with a HelperDependent (Gutted) Adenovirus Encoding Murine Dystrophin Renald Gilbert,1 Roy W R Dudley,2 An-Bang Liu,3 Basil J Petrof,2 Josephine Nalbantoglu,4 George Karpati.4 Genomics and Gene Therapy Vector Group, Biotechnology Research Institute, NRC, Montreal, QC, Canada; 2Respiratory Division, McGill University, Montreal, QC, Canada; 3Department of Neurology, Tzu Chi Medical Center, Hualien, Taiwan; Neuromuscular Research Group, Montreal Neurological Institute, Montreal, QC, Canada Dystrophin gene transfer using helper-dependent adenoviruses (HDAd), which are deleted of all viral genes, is a promising option to treat muscles in Duchenne muscular dystrophy We investigated the benefits of this approach by injecting the tibialis anterior (TA) muscle of neonatal and adult dystrophin-deficient (mdx) mice with a fully deleted HDAd (HDCBDysM) This vector encoded two full-length murine dystrophin cDNAs regulated by the powerful cytomegalovirus enhancer/β-actin promoter At 10 days postinjection of neonatal muscles, 712 fibers (42% of the total number of TA fibers) were dystrophin positive (dys+), a value that did not decrease for months (the study duration) In treated adults, maximal transduction occurred at 30 days post-injection (414 dys+ fibers, 24% of the total number of TA fibers), but decreased by 51% after months All studied aspects of the pathology were improved in neonatally-treated muscles: the percentage of dys+ fibers with centrally localized myonuclei remained low, localization of the dystrophin associated protein complex was restored at the plasma membrane, muscle hypertrophy was reduced, maximal force generating-capacity and resistance to contraction-induced injuries were increased The same pathological aspects were improved in the treated adults, except for reduction of muscle hypertrophy and maximal force generating capacity We demonstrated a strong humoral response against murine dystrophin in both animal groups, but mild inflammatory response occurred only in the treated adults Our data indicate that HDCBDysM is one of the most promising and efficient vectors for treating DMD by gene therapy, and that early muscle treatment using this vector would mitigate the DMD pathology more efficiently 252 Gel-Based Delivery of Recombinant AAV Vectors to Adult Murine Diaphragm Thomas J Fraites, Jr.,1 Cathryn Mah,1 Irene Zolotukhin,1 Barry J Byrne.1 Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States The diaphragm and respiratory muscles are important targets for gene transfer and therapy for a host of dystrophies and myopathies Transgenic and knockout mice provide the most widely available and genetically homogenous models in which to test potential therapies The murine diaphragm, which is six to eight cells thick in most normal mice, presents unique delivery challenges We have sought to develop methods for efficient and uniform delivery of recombinant adeno-associated virus (rAAV) vectors to mouse diaphragm We constructed rAAV vectors based on rAAV serotypes 1, 2, and 5, and evaluated their utility for diaphragmatic gene delivery with and without a gel-based delivery vehicle Recombinant AAV2 vector plasmids encoding the cytomegalovirus immediate-early promoterdriven beta galactosidase reporter gene were cross-packaged into AAV1, 2, and capsids as previously described (Zolotukhin, et al., 2002 Methods 28(2):158-67) Vectors were mixed at room S99 GENE THERAPY OF MUSCLE DISEASES temperature with a water-soluble, bacteriostatic, gelatin-based gel and directly applied to the abdominal surface of the diaphragm Free virus, without vehicle, was also directly applied to control diaphragms All vectors were administered at a dose of 5x109 particles Four weeks after gene delivery, diaphragm tissues were harvested and assessed for enzyme activity by X-gal staining B.J.B and the University of Florida may be entitled to patent royalties for inventions described in this abstract 253 Widespread Gene Expression of dsarcoglycan in the Bio14.6 Dystrophic Hamster Hanidleg Muscles by Pressurized Delivery of a Double-Stranded AAV Vector Tong Zhu,1 Liqiao Zhou,1 Bing Wang,1 Juan Li,1 Xiao Xiao.1 Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA Introduction: Gene transfer of the missing d-sarcoglycan in the limb girdle muscular dystrophy hamster Bio14.6 by AAV vectors is an effective treatment But systemic gene delivery through blood vessel and transduction efficiency of AAV vectors need further improvement Here we tested the pressurized intra-arterial injection method to transfer a novel double-stranded AAV vector carrying the d-sarcoglycan gene into the hindlimb muscle of Bio14.6 hamsters Method: To deliver the AAV vector through artery into a large group of the muscles, the femoral vessels were carefully dissected under a surgical microscope Two overlapping rubber tourniquets were transmuscularly placed at the level of the proximal thigh A microvascular clamp was placed to temporarily occlude the femoral vessels A 32G intracranial catheter was canulated distally into the femoral artery After the tourniquets were tightened , 4x1012 particles of dsAAV-CMV-d-sarcoglycan diluted in ml PBS was injected into the artery as fast as possible (normally in 8-10s) Local Intramuscular injection was also performed as a positive control with 2x1012 AAV vector particles into the gastrocnemius and tibialis anterior muscles, respectively All animals were sacrificed or months after injection Cryosections of Quadriceps, GAS and TA muscles were obtained for anit-d-sarcoglycan immunostaining Results: The untreated muscle cells showed a degeneration morphology without sarcoglycan expression; In contrast, in the intravascularly treated hindlimb, sustained and uniform expression of sarcoglycan was observed on the cell membranes of all muscle groups downstream of the vasculature However, in local intramuscular injection group, the sarcoglycan only expressed locally on the membrane of muscle cells in the injected muscle Uneven expression of the d-sarcoglycan was observed In general, no toxicity was observed for both gene delivery methods Conclusion: The pressurized intravascular injection method can systemically deliver the dsAAV-CMV-d-sarcoglycan to the hindlimb muscles, which offers potential clinical significance for future gene therapy trials S100 254 AAV Mediated Co-Delivery of Igf-I and Dystrophin to mdx Mouse Muscle Simone Abmayr,1 Tara J McNair,1 Rob W Crawford,1 Jeffrey S Chamberlain.1 Department of Neurology, University of Washington School of Medicine, Seattle, WA Gene replacement therapy for muscular dystrophy has been shown to improve morphological features and functional properties of dystrophic mdx mouse muscle However, viral vector mediated delivery of dystrophin to all muscle fibers remains a challenging goal Insulin-like growth factor-I (Igf-I) was found to enhance muscle regeneration and to maintain muscle mass and function in old and dystrophic animals Igf-I is a secreted polypeptide and can therefore target virus infected and non-infected cells by binding to its receptor and triggering proliferative and differentiation responses and antiapoptotic pathways Our goal is to co-deliver dystrophin and IgfI to dystrophic muscle to determine if the protective effect of Igf-I is synergistic with the beneficial effects of dystrophin in ameliorating the mdx phenotype We have cloned and characterized the isoforms of Igf-I that are expressed in normal and dystrophic mouse muscle Based on this information, we have generated several AAV vectors that express the major muscle isoform of Igf-I Our AAV vectors express Igf-I under the control of the CMV and the muscle specific promoters, CK6 and desmin We have developed a real time PCR assay to quantify RNA expression and to characterize the relative strength of the promoters Intramuscular injection of these AAV Igf-I vectors into C57/BL10 mice leads to levels of Igf-I mRNA expression up to 500-fold above normal Currently, we are investigating the effects of this overexpression on the muscle function and morphology Additionally, we have co-injected AAV carrying micro-dystrophin with AAV carrying Igf-I into dystrophic muscle and are studying their relative and combined potential for reversing the dystrophic pathology of the mdx mouse 255 Local Delivery of VEGF165 by AAV Vectors Protects Skeletal Muscle from Injury and Promotes Muscle Regeneration Nikola Arsic,1 Serena Zacchigna,1 Lorena Zentilin,1 Genaro Ramirez-Correa,1 Sabrina Tafuro,1 Lucia Pattarini,1 Alessandro Salvi,2 Gianfranco Sinagra,2 Mauro Giacca.1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 2Cardiology Unit, Ospedale Maggiore, Trieste, Italy Vascular endothelial growth factor (VEGF) is a main regulator of blood vessel formation during embryogenesis and a potent inducer of neovascularization during adult life Recent evidence suggests that VEGF activity is not strictly specific for endothelial cells, but is also exerted on other cell types Here, we report on the role of VEGF165 in promoting myogenic precursor cell differentiation to form multinucleated myotubes in vitro, as well as in the enhancement of muscle regeneration in vivo By immunofluorescence on cultured satellite cell-derived primary myoblasts, we showed that VEGFR2 is strongly upregulated after days of culture under differentiating conditions, and remains highly expressed until the last stages of the differentiation process Similar results were obtained using the C2C12 myogenic cell line In both primary myoblasts and C2C12 cells VEGF determined cell cycle arrest and protected cells from apoptotic death Moreover, the administration of recombinant VEGF during C2C12 differentiation resulted in a significant increase in the number and in the length of the newly formed myotubes, resulting from myoblast fusion The effects of VEGF on muscle cell survival and regeneration in vivo were assessed by the injection of a high titer preparation of an Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy GENE THERAPY OF MUSCLE DISEASES AAV vector expressing VEGF in two established murine models of muscle damage Muscle fiber injury was obtained by injection of either 50% glycerol or mM cardiotoxin, which both induce rapid destruction of muscle fibers and strong inflammatory reaction The former treatment also recapitulates some of the hallmarks that define the physiopathology of Duchenne muscular dystrophy By immunohistochemistry we observed that injury with both glycerol and cardiotoxin induced expression of VEGFR2 in muscle fibers Delivery of AAV-VEGF resulted in a remarkable improvement in the preservation of viable fibers and in the induction of fiber regeneration at day 20 after damage with both agents Preservation of tissue architecture was almost complete after injury with glycerol This effect involved a marked reduction in fiber apoptosis (as detected by reactivity to anti-caspase-3 antibody) and an increase in the number of regenerating myofibers Activity of AAV-VEGF strictly correlated with the dose of vector administered These results demonstrate that VEGF exerts a powerful and specific effect on muscle cell survival and myogenic differentiation This conclusion implicates that gene delivery of VEGF, besides induction of therapeutic angiogenesis, might be considered for the induction of muscle regeneration for the treatment of a variety of muscular disorders 256 Rapid Identification of Novel Canine Models of Duchenne Muscular Dystrophy Bruce F Smith,1 Glenn E Morris,2 Joe N Kornegay,3 Richard J Bartlett.4 Scott-Ritchey Research Center, Auburn University, Auburn, AL, United States; 2NEWI, University of Wales, Wrexham, United Kingdom; 3College of Veterinary Medicine, University of Missouri, Columbia, MO, United States; 4NIAMS, National Institutes of Health, Bethesda, MD, United States Duchenne muscular dystrophy (DMD) is an X-linked, progressive muscle wasting disease with fatal consequences, which is caused by mutations in the human dystrophin gene DMD presents unique challenges to gene therapy, due to the size of the gene and resulting cDNA and the wide variety and complexity of the mutations involved As an X-linked recessive disease, new mutations present themselves at higher rates than in autosomally inherited diseases In addition, mutations in DMD differ from those classically seen in inherited disease, with an emphasis on deletions Animal models for DMD have been described in mice, cats and dogs with mutations in the respective dystrophin genes, located on the xchromosome Both cats and mice have less severe forms of the disease In the murine model, additional mutations such as a utrophin gene knockout must be bred into the mdx background to reproduce the pathology, symptomatology and fatal consequences found in DMD patients Only in dogs does the disease carry the full spectrum of clinical phenotypes found in patients with DMD, including the fatal consequences of muscle wasting Thus, only canine dystrophinopathies recapitulate the human disease with a single gene mutation In dogs and humans the structure, size and exon junction location of the dystrophin genes are remarkably similar Thus, it is not surprising that the mutations in the animal models described thus far prove to be different mutations Any therapy developed for treatment of this spectrum of mutations must be capable of overcoming the consequential spectrum of clinical conditions To maximize the potential for pre-clinical evaluation of potential therapies, a spectrum of mutations in canine breeds with the consequential continuum of clinical phenotypes would prove invaluable To date, canine models have been described in the Golden Retriever, Rottweiler, and German Short Haired Pointer breeds We have developed a program to rapidly screen possible new canine models of dystrophin deficiency using a combination of specific antibodies and rapid, directed sequencing In the initial Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy screen, antibody binding data is used to identify the likely portion of the molecule affected by the mutation This region is then amplified by RT-PCR in one kilobase sections and sequenced Data from these PCR reactions also provides an indication of small deletions or insertions, either due to the failure of the PCR reaction to amplify, or due to alterations in amplicon size Analysis of the cDNA sequence data allows the specific mutation to be identified or it may indicate possible genomic alterations that affect cDNA structure This approach allows rapid identification of the mutation in the model, allowing the suitability of a model to be determined without significant investment or delay By determining the mutation in additional dogs that have presented with clinical muscular dystrophy, a spectrum of single gene mutations in canine models can be described that will allow more appropriate screening of therapeutic approaches than is possible in inbred mice Data from the analysis of new canine models of Duchenne Muscular Dystrophy will be presented 257 Muscle Stem Cells Promote Nerve Regeneration and Contribute to the Development of Neuronal Tissues Zhuqing Qu-Petersen,1 James Cummins,1 Aiping Lu,1 Arvydas Usas,1 Ron Jankowski,1 Makoto Ikezawa,1 Ryosuke Kuroda,1 William de Groat,2 Johnny Huard.1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Pharmacology, University of Pittsburgh, Pittsburgh, PA Muscle-derived stem cells (MDSC) isolated from normal neonatal mouse skeletal muscle via the preplate technique display an improved transplantation capacity when implanted in the skeletal muscle of dystrophin-deficient mdx mice The benefits associated with MDSC transplantation are at least partially attributable to their impressive self-renewal ability and their capacity to undergo multipotent differentiation to form myofibers, blood vessels, and peripheral nerves in the injected muscle We investigated whether the injection of normal MDSC clones can promote the regeneration and repair of 3-mm sciatic nerve defects created in mdx mice We found that MDSC in the nerve defect area can differentiate into Schwann cells that significantly promote axonal regeneration and subsequently alleviate muscle atrophy in denervated gastrocnemius muscles The regenerating capacity of the MDSCs in the nerve defect appears to be influenced by the time at which MDSC injection is performed post-injury Normal MDSC also contribute to the formation of astrocytes and neurons in the hippocampus and cerebral cortex following either intracranial transplantation or intravenous dissemination of the cells These findings demonstrate that MDSC possess remarkable plasticity in response to environmental cues and suggest the potential promise of MDSC-based cell therapies for treatment of various neuromuscular diseases 258 AAV Vector-Mediated Canine MiniDystrophin Gene Expression in mdx Mice Bing Wang,1 Mengnan Tian,1 Chunping Qiao,1 Tong Zhu,1 Juan Li,1 Xiao Xiao.1,2 Dept.of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States; 2Dept of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, United States Duchenne muscular dystrophy (DMD) is the most common disabling and lethal genetic muscle disorder, affecting one of every 3,500 males No effective treatment is currently available for DMD The mdx mouse has been the most widely used animal model for DMD, although mdx lacks major clinical deterioration seen in human patients In contrast, the golden retriever muscular dystrophy (GRMD) dog, as a large animal model, displays remarkable clinical and pathological similarities to the human DMD patients Therefore, the GRMD is well conceived as a clinically more relevant S101 GENE THERAPY OF MUSCLE DISEASES DMD model Previously, we have attempted AAV-mediated human mini-dystrophin gene delivery to treat the GRMD, but the result was not striking We believe that the human dystrophin gene expression in dog may have elicited immune-response in the dystrophic dog In this report, we have cloned the dog dystrophin cDNA via RTPCR from normal dog muscle and generated a dog version minidystrophin gene (3.8 kb, including rods) that can be readily package into AAV vector along with CMV promoter We show that the dog mini-dystrophin can be expressed in mdx mice at high levels at one month after vector injection Immunostaining of the consecutive sections, using antibody against N-terminus of human dystrophin, revealed the restoration of the missing dystrophin onto the plasma membrane H & E staining also displayed normal histology and the lack of fibrosis and infiltration in the vector transduced area The results prove the feasibility of using AAV dog mini-dystrophin vector in the large GRMD canine model 259 Lentiviral Vector Mediated Gene Transfer to Mouse Skeletal Muscle Cells: Potential Applications for Duchenne Muscular Dystrophy Sheng Li,1 En Kimura,1 R W Crawford,1 B Fall,1 J M Scott,1 J C Angello,2 R Welikson,2 S D Hauschka,2 J S Chamberlain.1 Department of Neurology, University of Washington School of Medicine, Seattle, WA; 2Department of Biochemistry, University of Washington School of Medicine, Seattle, WA Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD) We have shown previously that delivery of mini- or full-length dystrophin genes to muscles of mdx mice, a model of DMD, can prevent and partially reverse the dystrophic pathology However, gene therapy for DMD will require systemic delivery, and sustained expression, of therapeutic dystrophins in widely distributed skeletal muscles Lentiviral vectors have a relatively large transgene carrying capacity and are able to integrate into non-dividing cells We explored the use of lentiviral vectors for transferring genes into mouse skeletal muscle cells in vitro and in vivo The lentiviral vectors efficiently transduced both proliferating and terminally differentiated mdx muscle cells in vitro, and transgeneexpressing myoblasts were able to differentiate normally without any obvious toxicity We demonstrated that even a small version of the murine creatine kinase regulatory cassette maintained musclespecific activity in lentivirally-transduced cells We were also able to transduce with high efficiency the minidystrophin-lentivirus cassettes into a variety of other cell types in vitro, including myoblasts derived from dystrophic dogs and hematopoietic stem cells Although we were able to obtain moderate levels of skeletal muscle transduction in vivo by direct intramuscular injection, the relatively low titer of lentiviral preparations combined with physical barriers to virus diffusion will limit the direct application of lentiviral vectors for transferring therapeutic genes into muscles Nonetheless, the ability to successfully transduce both muscle and bone marrow cells in vitro with mini-dystrophin expressing lentiviral vectors suggests that this system may have great potential for developing ex vivo cell therapies for DMD S102 260 Functional Analysis of Dystrophin in Vascular Smooth Muscle Cells in Duchenne Muscular Dsytrophy Kaori Ito,1 Shigemi Kimura,1 Shiro Ozasa,1 Makoto Ikezawa,1 Misao Suzuki,2 Kowashi Yoshioka,1 Makoto Matsukura,1 Takashi Hiranuma,1 Takeshi Miwa,3 Teruhisa Miike.1 Department of Child Development, Kumamoto University Medical School, Kumamoto, Kumamoto, Japan; 2Division of Transgenic Technology Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Kumamoto, Japan; 3Department of Oncogene Research, Research Insutitute for Microbial Deseases, Osaka University, Suita, Osaka, Japan Duchenne muscular dystrophy (DMD) is an X-linked fatal disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin Dystrophin is a membrane-associated protein that provides a link in a chain of proteins between the actin cytoskeleton to extracellular matrix and comprises a dystrophin-glycoprotein complex (DGC) Dispite the abundance of new information on these molecules, there currently is no effective treatment for DMD because the mechanism by which dystrophin deficiency produces the clinical phenotype is poorly understood Two principal theories have been proposed to explain the pathogenesis of DMD The first is that dystrophin deficiency destabilizes the sarcolemmal integrity, thereby rendering the muscle fibers susceptible to damage during contractions The second theory is that disruption of DGC gives rise to the reduction of the scaffolding function that recruits signaling proteins such as neuronal nitric oxyde synthase (nNOS) to the membrane Recent studies indicate that nNOS in skeletal muscle plays a key role in the regulation of the blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to alphaadrenergic receptor activation This protective mechanism is defective in both, DMD patients and mdx mice, an animal model of DMD We hypothesized that dystrophin deficiency also causes the reduction of nNOS in vascular smooth muscle cells (VSMCs), leads to vascular dysfunction and exacerbates muscle pathology We therefore generated transgenic mice expressing 14Kb full-length human dystrophin cDNA under the transcriptional control of the smooth muscle alpha-actin promoter These mice were then crossed with mdx mice, resulting in three independent SMTg/mdx lines which harbor the dystrophin gene only in SMCs PCR and southern blot analysis were performed to verify founders and stable transgenic lines The expression pattern was detectable by semi-quantitative RT-PCR analysis and immunohistochemical staining, which showed the specific expression of transgene in SMCs We also report the histological characteristics of SMTg/mdx mice such as central nucleation, fiber size variability, and CK concentrations as compared to C57BL/10 control mice and mdx mice We believe that our SMTg/ mdx mouse model is worth exploring to gain a better understanding of the functon of dystrophin in VSMCs and the pathophysiology of DMD patients We also believe that the introduction of dystrophin gene into VSMCs is necessary for the effective treatment for DMD References 1) Kobzik L., et al Nature 1994; 8(372):546-48 2) Brenman JE., et al.Cell 1995; 82(5):743-52 3) Chang WJ., et al Proc Natl Acad Sci USA 1996; 93(17):9142-47 4) Gail D.Thomas., et al Proc Natl Acad Sci USA 1998; 95:15090-95 5) Mikael Sander, et al Proc Natl Acad Sci USA 2000; 97(25):13818-23 6) Michelle Wehling, et al J Cell Biol 2001; 155(1):123-31 Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy GENE THERAPY OF MUSCLE DISEASES 261 An AAV Vector-Mediated Micro-Dystrophin Expression Ameliorates Dystrophic Phenotypes of mdx Muscles Miki Sakamoto,1 Madoka Yoshimura,1 Katsutoshi Yuasa,1 Toshifumi Yokota,1 Takaaki Ikemoto,2 Xiao Xiao,3 Yuko Miyagoe-Suzuki,1 Shin’ichi Takeda.1 Molecular Therapy, National Institute of Neuroscience, Kodaira, Tokyo, Japan; 2Pharmacology, Saitama Medical School, Moroyama, Saitama, Japan; 3Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by a defect in the dystrophin gene, and characterized by progressive muscle weakness, cardiomyopathy and early death An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches for the treatment of DMD, but it has a limitation in insertion size up to 4.9 kb Therefore, a full-length dystrophin cDNA (14 kb) cannot be incorporated into an AAV vector To find a short but functional dystrophin cDNA, we generated a series of rod-truncated micro-dystrophin (M3, 3.7 kb; AX11, 4.4 kb; CS1, 4.9 kb), and generated transgenic (Tg) dystrophin-deficient mdx mice expressing each of microdystrophins CS1 Tg mdx mice showed lowest levels of serum creatine kinase, complete amelioration of muscle pathology, and nearly full restoration of contractile force (Biochem Biophys Res Commun 293:1265, 2002) To test whether AAV vector-mediated CS1 micro-dystrophin expression can ameliorate the dystrophic phenotypes of mdx muscle, we constructed an AAV vector expressing micro-dystrophin CS1 We used skeletal muscle-specific MCK promoter to drive the CS1 gene, since the MCK promoter in AAV vector drives longer expression of the LacZ gene than the CMV promoter in skeletal muscle (Gene Ther 23:1576, 2002) To reduce the length of CS1 cDNA, we deleted 5’- and 3’-untranslated regions and the coding region corresponding to exons 71- 78 (ΔCS1, 3.8 kb) To evaluate the therapeutic effects of AAV-MCKΔCS1, we first injected the AAV vector into anterior tibial (TA) muscles of 10-dayold mdx mice At this age,mdx mice show no signs of muscle degeneration Therefore, it is easy to evaluate the therapeutic effects of the vector by counting the ratio of centrally nucleated myofibers We then injected the vector into 5-week-old mdx mice whose muscles show active cycles of muscle degeneration/regeneration When the AAV vector was introduced into 10-day-old mdx mice, the expression of micro-dystrophin continued for a long time, but dystrophinpositive fibers scattered; 10 to 23% at 24 weeks after the AAV injection H&E staining of muscle tissues showed nearly normal morphology of dystrophin-positive fibers In contrast, extensive expression of micro-dystrophin was achieved when 5-week-old mdx muscles were treated At weeks after the AAV vector injection, a large percentage of fibers were dystrophin-positive (10 to 50%) Even 24 weeks after the injection, 15 to 75 % of myofibers expressed micro-dystrophin Dystrophin-positive fibers often had centrally located nuclei, however, ratio of these fibers was significantly reduced compared with that of dystrophin-negative fibers We then isolated AAV-MCKΔCS1-treated and non-treated mdx TA muscle and measured tetanic force Non-treated mdx muscle showed reduced specific tetanic force, but AAV-injected mdx TA muscles showed moderate improvement of the specific force Thus, our study demonstrated that ΔCS1 micro-dystrophin introduced by an AAV vector both before the onset of dystrophic changes and during ongoing muscle degeneration successfully protected mdx muscle Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy 262 Chronic Inflammation-Induced Extrasynaptic Utrophin Upregulation in Muscle Fibers of Immune Competent mdx Mice Is Related to Reduced Calpain Activity of Muscle Ishrat Waheed,1 Renald Gilbert,1 Basil Petrof,2 Josephine Nalbantoglu,1 George Karpati.1 MNI; 2RVH Chronic inflammation induced in the anterior tibialis muscles of immune competent mdx mice by intramuscular injection of a first generation adenovirus (FGAV) with strong beta galactosidase expression produces, by 30 days, appreciable amounts of extrasynaptic utrophin (utr) Utr is close structural and functional analogue of dystrophin (dys), and the amount of the extrasynaptic utr produced by chronic inflammation is sufficient to mitigate the deleterious effects of dys deficiency (reduced muscle fiber necrosis, restoration of dys-associated proteins, force generation impairment, etc.) Certain proinflammatory cytokines have been suspected to have a role in this process which, among other things, is supported by our finding that in certain types of immune incompetent mice (i.e TNF-alpha gene-ablated), the above-described extrasynaptic utr upregulation does not occur By contrast, in the IL-6 gene-ablated animals, utr levels remain unaffected, possibly because of vigorous compensation by IL-6 analogues Here we also report that activity of calpain was significantly reduced in the inflammatory mdx muscles compared to non-inflammatory mdx controls (p