ABSTRACTS THAT WILL BE PRESENTED IN THE 8TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF GENE THERAPY, JUNE 1-5, ST LOUIS, MISSOURI

1) Antisense Therapy for Duchenne Muscular Dystrophy: A Realistic Possibility

Qi L. Lu, Adam Rabinowitz, HaiFang Yin, Julia Alter, George Bou-Gharios, Terrence Partridge. Muscular Dystrophy Laboratory, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC; Muscle Cell Biology, MRC Clinical Science Centre, London, United Kingdom; Department of Renal Medicine, Imperial College, Hammersmith Hospital Campus, London, United Kingdom

Duchenne Muscular Dystrophy (DMD) is the most common form of muscular dystrophy affecting 1 in every 3500 live male births. The disease is characterized by severe muscle wasting and weakness, which becomes clinically evident between the ages of 3 to 5 years. The milder form of the disease is Becker muscular dystrophy (BMD) with a spectrum of phenotypes ranging from almost asymptomatic to mild forms of DMD. Both DMD and BMD are the results of mutations in the dystrophin gene. The phenotypic distinction between DMD/BMD can be explained by the reading-frame theory where DMD is caused by frame-shifting or nonsense mutations leading to premature termination of protein synthesis whereas BMD is caused by mutations resulting in altered, but in-frame transcripts. One promising alternative approach for treatment of DMD is antisense oligonucleotide (AO)-mediated gene correction at RNA level (antisense therapy). This technique uses small oligonucleotides to correct frame-shifting or nonsense mutations by skipping a mutated exon or exons which disrupt the reading frame in such a way that restores reading frame of the dystrophin transcript. This results in the expression of a truncated, but at least partially functional dystrophin. Antisense therapy is uniquely applicable to DMD treatment for many reasons. First, the gene consists of 79 exons and muscle form of dystrophin protein can be divided into amino terminal, rod, cysteine-rich and carboxy terminal domains. However, the rod domain of the dystrophin gene appears not to be critical for its functions. Second, the majority of DMD mutations occur within this non-critical region of the dystrophin gene. Thus correction of frame-shifting mutations by skipping the mutated exon or other exons necessary for restoration of reading frame will retain critical functions of the protein. We have demonstrated that specifically designed 2-O-methyl phosphorothioate AO (20MeAO) delivered by intramuscular injections was able to effectively skip the mutated region (exon) of the dystrophin gene in animal model of DMD with functional improvement in the muscles. Dystrophin induced by 20MeAO remains at detectable levels even 3 months after intramuscular injections. This instigated clinical trials in several countries. We now demonstrated that the AOs delivered by simple intra-venous injections can induce dystrophin expression in body-wide skeletal muscles up to therapeutic levels. The simplicity and safety of the antisense therapy provide a realistic possibility for treatment of the majority of DMD mutations.
 

2) Ex Vivo Gene Therapy for Duchenne Muscular Dystrophy: Lentiviral and PhiC31 Integrase Approaches

Simon P. Quenneville, Pierre Chapdelaine, Joël Rousseau, Michele P. Calos, Jacques P. Tremblay. Genetique Humaine, CRCHUL, Sainte-Foy, QC, Canada; Department of Genetics, Stanford University School of Medecine, Stanford, CA

Duchenne muscular dystrophy is the most severe muscular dystrophy. It is caused by the absence of dystrophin in muscle fibers. This absence lead to increased muscle damage, loss of muscle mass, loss of strength, respiratory and heart failure. This disease as currently no treatment. Myogenic cells transplantation is a possible cure for Duchenne muscular dystrophy. However, allogeneic graft success relies on the use of efficient but toxic immunosuppressive drugs. The use of these drugs is a major problem that could be solved by the use of ex vivo gene therapy. This method consists in genetically modifying patient myoblasts before their auto-transplantation. In this study, a viral and a non viral method were tested to perform the genetic modification. The co-transfection (nucleofection) of a PhiC31 integrase and a dystrophin expressing plasmid as already led to a stable expression of the full length dystrophin¹. This was the biggest expression cassette ever stabilized in primary cultured human myoblasts. Using this method, we are now capable of transferring transgene expressing cells into a muscle, leading to expression into the muscle fibers. However, this method is not very efficient. This problem could be solved using viral vectors. We have generated eGFP and eGFP-micro-dystrophin expressing lentiviral vectors under the control of the CMV and MCK promoters. Following in vitro infection of human myoblasts, the cells were engrafted into SCID mice muscles. We were able to detect the expression of both transgenes into these muscle fibers one month after the engraftment. Evans bleu will be injected in the blood of the mice before an eccentric exercise. We will verify whether the expression of dystrophin in the muscle fibers reduces their damage during the exercise and thus the incorporation of Evans blue. This work indicates that ex vivo gene therapy is a possible approach to treat Duchenne muscular dystrophy.
1-Quenneville SP et al., Mol Ther. 2004 Oct;10(4):679-87.
 

3) Immunity to AAV-Mediated Gene Therapy in a Random-Bred Canine Model of Duchenne Muscular Dystrophy

Zejing Wang, Michael J. Blankinship, Paul Gregorevic, Marie-Terese Little, Rainer J. Storb, James M. Allen, Stephen J. Tapscott, Jeffrey S. Chamberlain, Christian S. Kuhr. Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Neurology, University of Washington, Seattle, WA

Introduction: Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Studies in the mdx mouse model of DMD have shown that muscle membrane integrity and function can be improved by AAV-mediated delivery of a functional dystrophin protein. To assess the potential clinical utility of treating human DMD patients with AAV-mediated gene delivery, we performed a series of direct intra-muscular injections in random-bred normal dogs and in dogs with muscular dystrophy caused by a dystrophin mutation (xmd dogs). Methods: AAV serotypes 2 and 6 carrying different promoter-transgene cassettes were produced as previously described for the murine studies and purified either on a heparin column or with a combination of heparin column and cesium chloride gradient. Direct intramuscular injections of virus (10¹², 10¹⁰, or 10⁸ viral genomes per site) in a total volume of 250 ul were performed. The injection sites were biopsied under anesthesia between 2 and 12 weeks after injection. Results: Injection of either AAV6 or AAV2 expressing CMV-b-galactosidase (b-gal) induced a strong inflammatory response containing both CD8+ and CD4+ T-lymphocytes with peak tissue destruction 4 weeks following viral injection. A similar robust immune response was seen with injection of AAV6-RSV-alkaline-phosphatase, AAV6-CMV-canine-factor-IX (cFIX) and with empty AAV6 capsid alone. Additional purification of the AAV6-CMV-cFIX by centrifugation through a cesium chloride gradient did not diminish the immune response. Continuous immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) largely prevented the immune response to AAV6-CMV-b-gal in a normal dog for up to four weeks and permitted robust transgene expression. The same immunosuppressive regimen did not prevent an immune response to AAV6-CMV-cFIX or AAV6-CMV-human-micro-dystrophin in an xmd dog, suggesting that a more aggressive immunosuppressive regimen might be necessary in the xmd dog. Conclusions: Taken together, our results suggest that AAV capsid proteins, or proteins associated with the capsid, elicit significant immune responses when directly injected into skeletal muscle of normal random-bred dogs. The combination of CSP and MMF effectively prevents the immune responses in a normal dog, but not in an xmd dog, possibly due to the pre-existing inflammatory nature of the DMD muscle disease or due to genetic variation in a random-bred animal population. The robust immune responses to AAV2 and AAV6 in random-bred dogs contrast with the lack of an immune response in studies by others in inbred mice and dogs. Further studies will be necessary to determine the nature of the immune responses and to develop appropriate immunosuppressive regimen for AAV gene delivery to xmd muscle.
 

4) Stable Genome Alteration of the Dystrophin Gene for the Treatment of Duchenne Muscular Dystrophy (DMD) Due to Frame-Shift Mutations Using Oligonucleotide-Mediated Gene Editing

Carmen Bertoni, Thomas A. Rando. Neurology, Stanford Universdity School of Medicine, Stanford, CA

Duchenne muscular dystrophy is an X linked neuromuscular disorder characterized by lack of dystrophin gene expression. Mutations in the dystrophin gene include single point mutations, large deletions and frame shift mutations. Genome editing of the dystrophin gene represents an attractive approach to restoration of dystrophin into skeletal muscle of DMD patients. Oligonucleotide-mediated single base substitution has the potential to completely correct the mutation in the case of single point mutations, or to restore the reading frame of the dystrophin gene to correct frame shift mutations. Furthermore oligonucleotide-mediated gene correction allows the gene to remain under the control of its own regulatory mechanisms.
We have tested the applicability of genome editing of the dystrophin gene in the mdx mouse model for DMD. This strain contains a stop codon in exon 23 of the dystrophin gene that is responsible for the absence of dystrophin protein in skeletal muscles. As a target for the single base substitution we have chosen the splice boundary of exon 23 of the mouse dystrophin gene in order to induced exon skipping to bypass the nonsense mutation and induce expression of internally deleted but functional dystrophin proteins. We have designed single stranded oligonucleotides (ssODNs) complimentary to the coding or the non-coding strand of the donor site of exon 23. Modifications were inserted into the oligonucleotides in an attempt to recruit specific repair processes present in eukaryotic cells and increase the efficiency of the repair process. For these kinds of studies, muscle cells represent an very appealing system since the repair processes involved can be characterized in dividing cells (proliferating muscle precursor cells) as well as mature myofibers at the terminally differentiated stage, thus providing further insight in the process that regulate gene correction.
CpG modifications were introduced at the 3 end of ssODNs as well as at the targeted base. The ability of these modified ssODNs to increase gene repair was studied in muscle cells both in vitro and in vivo. Correction of the dystrophin gene was shown to occur at the genomic level using all types of oligonucleotides and was shown to be stable over prolonged periods of time. In muscle cells in culture, restoration of dystrophin expression was analyzed at the protein level by western blot and immunostaining analysis, and at the mRNA level by RT-PCR. In vivo analysis also showed restoration of dystrophin in skeletal muscles of injected mice by immunostaining. These studies confirm the applicability of genome editing in cases in which DMD is due to single point and frame-shift mutations.
 

5) Embryonic Stem Cell-Mediated Regenerative Therapy for Duchenne Muscular Dystrophy

Shiro Ozasa, Shigemi Kimura, Makoto Matsukura, Kaori Ito, Makoto Ikezawa, Hiroe Kawano, Teruhisa Miike, Kenichi Yamamura, Kimi Araki, Kuniya Abe Hitoshi Niwa. Department of Child Development, Kumamoto University Graduate School, Kumamoto, Japan; Institute of Molecular Embryology and Genetics, Kumamoto University Graduate School, Kumamoto, Japan; Research & Development Team for Mammalian Cellular Dynamics, BioResource Center, RIKEN Tsukuba Institute, Tsukuba, Ibaragi, Japan; Laboratory of Pluripotent Cell Studies, RIKEN center for developmental Biology, Kobe, Hyogo, Japan

Introduction
Duchenne muscular dystrophy (DMD) is a severe muscle degenerative disorder caused by mutations in the dystrophin gene. Embryonic stem cell (ES cell) transplantation is one of the more promising therapies because ES cells are available in large quantities and can serve to systemically restore affected muscles of DMD patients. Although ES cells have shown to be capable of differentiating into various tissues and cell types, it has been difficult to induce differentiation of ES cells specifically into muscle fibers only. We are proposing a method to overcome this impediment by establishing genetically engineered ES cells which harbor a tetracycline-regulated expression vector encoding the myogenic transcriptional factor MyoD.
Methods and Results
ZHTc6 is a mouse-derived ES cell line in which Tet-Off System is integrated. ZHTc6 can be induced to express the POU transcriptional factor Oct-3/4 on the removal of doxycycline, a derivative of tetracycline. We cultured ZHTc6 without feeders in LIF-supplemented medium. We constructed Supertargeting Vector-MyoD, which is targeted upon the electroporation to invoke the homologous recombination and replace the inducible Oct-3/4 gene of ZHTc6 with MyoD gene. Southern-blot analysis revealed that three out of 6 clones were recombinants. The recombinants, hereinafter referred to as ZHTc6-MyoDs, were induced to differentiate nonspecifically in the differentiation medium in the absence of doxycycline. Western analysis of the differentiated cells revealed the expression of MyoD, desmin, myogenin and fast myosin heavy chain (fMHC). The involvement of MyoD in myotube formation was substantiated by colocalization of MyoD with the muscle-specific protein dystrophin as revealed by immunocytochemical analysis. Desmin and fMHC were also detected by immunocytochemical analysis. Serial flow cytometric analysis revealed that the undifferentiated ZHTc6-MyoDs initially expressed Sca1 and c-kit at a high level (more than 90%), but the expression gradually decreased during differentiation. To the contrary, only a small fraction (less than 1%) of ZHTc6-MyoDs expressed CD34 throughout the differentiation. We intramuscularly injected 2-day-differentiated ZHTc6-MyoDs into gastrocnemius muscles of adult mdx-nude mice. Seven weeks after injection, tumors developed in the injected area to the size of 20 mm in diameter. Examination of the tumor specimen uncovered the presence of the clusters of dystrophin-positive myofibers surrounded by granular tumor cells.
Conclusion
In this system, we can maintain ES cells undifferentiated in the presence of doxycycline and induce ES cells to undergo myogenic specification on the removal of doxycycline in vitro.
 

6) Development of rAAV-Based Skeletal and Cardiac Muscle Regulatory Cassettes for Gene Therapy of Duchenne Muscular Dystrophy

Maja Z. Salva, Charis L. Himeda, Phillip Tai, Michael J. Blankinship, Paul Gregorevic, James M. Allen, Leonard A. Meuse, Jeffrey S. Chamberlain, Stephen D. Hauschka. Bioengineering, Univeristy of Washington, Seattle, WA; Biochemistry, University of Washington, Seattle, WA; Neurology and Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington, Seattle, WA

A promising gene therapy approach for treatment of DMD involves systemic delivery of rAAV vectors encoding microdystrophin. This method was shown to mediate efficient transduction of all striated muscles and high-level transgene expression driven by the ubiquitous CMV promoter. However, the use of viral promoters increases the risk of an immune response, largely due to transgene expression in antigen-presenting cells, and may thus be inadequate for long-term therapy. An ideal regulatory cassette for gene therapy of DMD thus needs to direct high-level, tissue-specific expression in skeletal and cardiac muscle and be short enough to package into the rAAV microdystrophin construct.
Our lab has developed a series of muscle-specific regulatory cassettes based on the murine muscle creatine kinase (MCK) gene enhancer and promoter regions. The previous best small cassette, CK6 (580bp) drives microdystrophin expression in skeletal muscle following systemic rAAV delivery, at 10% of the level of the CMV promoter. However, expression in heart and diaphragm is very low. To improve expression, we combined the wild type MCK enhancer with a 190bp enhancer region from the murine alpha myosin heavy chain (MyHC) gene, which is expressed at high levels in cardiac muscle. Addition of the MyHC enhancer increased activity in MM14 skeletal myocytes by 5-fold as assessed by alkaline phosphatase reporter gene activity. Furthermore, high-level expression was detected in skeletal muscle, as well as heart and diaphragm after systemic delivery in mice of rAAV6 vectors encoding these constructs. The activity of the hybrid cassette was 7-fold higher in heart and 3-fold higher in diaphragm and soleus (predominantly slow-twitch muscle fibers) when compared to the wild type MCK cassette, while the activity in tibialis anterioris (predominantly fast-twitch muscle fibers) was unchanged. Expression was restricted to muscle tissue, as evidenced by nearly undetectable levels in the liver, spleen, lung, and aorta. Additional cassettes comprising the MyHC enhancer and various modifications of the minimal MCK cassette have also been tested in MM14 skeletal myocytes. The strongest cassettes were shown to possess 12-fold higher activity than the wild type MCK cassette, and were about 55% as active as the CMV promoter. These contained a 63bp deletion within the MCK enhancer, addition of a 50bp region downstream of the transcription initiation site, and a mutation that creates a consensus initiator-binding site from the terminal transferase promoter. We are currently testing activity of these constructs in mouse muscle and non-muscle tissues following systemic delivery of rAAV6 vectors. The strongest tissue-specific cassette will be evaluated for long-term expression of therapeutic levels of microdystrophin in the mdx mouse model for DMD.
 

7) Enhanced Plasmid-Mediated Dystrophin Expression in the mdx Mouse Model for Duchenne Muscular Dystrophy by a PhiC31 Integrase Plasmid System

Carmen Bertoni, Sohail Jarrahian, Thurman M. Wheeler, Yining Li, Eric C. Olivares, Michele P. Calos, Thomas A. Rando. Neurology, Stanford University School of Medicine, Stanford, CA; Genetics, Stanford University School of Medicine, Stanford, CA

Duchenne muscular dystrophy is caused by lack of dystrophin expression in skeletal muscles and is characterized by progressive degeneration of muscle fibers. To be effective, gene therapy approaches to DMD need to target a large number of fibers in the muscle, and the distribution of dystrophin through the fiber length needs to be sufficient to prevent fiber degeneration. Plasmid-based gene therapies have been shown to be a valid approach to the treatment of a variety of disorders including DMD. Concerns, however, have been raised about the ability of extrachromosomal vectors to sustain gene expression for prolonged periods of time at levels that are therapeutic.
We have tested the ability of the phiC31 integrase system to integrate plasmid vectors stably into the chromosomes of terminally differentiated myonuclei and maintain transgene expression over prolonged periods of time. Tibialis anterior muscles of wild-type animals were injected with a plasmid carrying a luciferase reporter gene under the control of the CK6 muscle specific promoter and a phiC31 integrase attB site. One group of muscles was co-injected with an equal amount of a CMV-driven integrase-expressing plasmid (pCSI) to direct site-specific integration, while contralateral muscles received empty vector (pCS). All muscles were subjected to electroporation to achieve a high level of plasmid delivery, and expression was followed using a bioluminescence live imaging system (BLIS). Shortly after delivery, the level of gene expression obtained in muscles that had received pCSI was 10-fold higher than in muscles receiving pCS. This increase in the gene expression level was maintained for up to 2 years after injection. Site-specific integration of the luciferase attB plasmid was confirmed by PCR analysis.
The beneficial effects of phiC31-mediated integration were also tested in the mdx mouse model for DMD. Muscles were co-injected with an attB plasmid carrying the luciferase reporter gene driven by the CK6 promoter and an attB plasmid carrying the full-length dystrophin gene, also driven by the CK6 promoter, in the presence of either pCS or pCSI. BLIS analysis showed persistent luciferase expression only in muscles co-injected with the integrase expression vector. The level of dystrophin expression was higher in muscles co-injected with pCSI, as demonstrated by RT-PCR analysis. Six months after gene delivery, the overall increase in dystrophin expression resulted in more than twice as many dystrophin-positive fibers in pCSI-injected muscles, compared to muscles injected with pCS. Distribution of dystrophin along the length of the fibers was also greater in muscle receiving the integrase plasmid. Plasmid integration was also confirmed in muscles co-injected with pCSI. CD4 and CD8 staining showed no increase in the inflammatory response in muscles that received integrase. Site-specific integration of plasmid DNA mediated by phiC31 integrase enhance plasmid-mediated gene expression and has a relevant clinical application for muscle diseases such as DMD.
 

8) Sustained Whole-Body Functional Rescue by Systemic Delivery of AAV8 Vectors in Heart Failure and Muscular Dystrophy Hamsters

Tong Zhu, Liqiao Zhou, Satsuki Mori, Zhong Wang, Charles Mctiernan, Chunping Qiao, Chunlian Chen, Daowen Wang, Juan Li, Xiao Xiao. Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA; Cardiovascular Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA; Cardiology, Tongji Hospital,Huazhong Science & Technology University, Wuhan, Hubei, China

The success of muscular dystrophy gene therapy requires widespread and stable gene delivery with minimal invasiveness. Using the delta-sarcoglycan (SG)-deficient hamster (TO-2), a congestive heart failure and muscular dystrophy hamster model, here we show that a single injection of double-stranded adeno-associated virus serotype 8 vector carrying human delta-sarcoglycan gene (dsAAV8-SG), without the need of any physical or pharmaceutical interventions, achieved nearly complete gene transfer and tissue-specific expression in the heart and skeletal muscles of the diseases hamsters.
Broad and sustained ( >12 months) restoration of the missing delta-sarcoglycan gene in the TO-2 hamsters corrected muscle cell membrane leakiness throughout the body, and normalized serum creatine kinase levels (an 50-100 fold drop). Histology examination revealed minimal or absence of central nucleation, fibrosis, and calcification in the skeletal muscle as well as the heart. Whole body functional analysis such as treadmill running showed dramatic improvement, similar to the wild-type F1B hamsters. Furthermore, cardiac function studies such as echocardiography revealed significantly increased %FS and decreased LVEDD and LVESD in the treated TO-2 hamsters. The survival time of treated TO-2 hamsters was dramatically prolonged.
To the summary, our study demonstrates that systemic AAV8 vector-mediated gene transfer could persistently ameliorate of cardiac and skeletal muscle pathology, profoundly improve cardiac and whole-body functions, and significantly prolong the life-span of the treated TO-2 hamsters. Future research is warranted to see if such profound gene delivery efficiency as well as therapeutic efficacy can be duplicated in large animals and eventually in human patients.
 

9) Dystrophin in Vascular Smooth Muscle Is Important for Duchenne Muscular Dystrophy Therapy

Kaori Ito, Shigemi Kimura, Gail D. Thomas, Shiro Ozasa, Makoto Matsukura, Makoto Ikezawa, Hiroe Ueno, Kowashi Yoshioka, Misao Suzuki, Takeshi Miwa Teruhisa Miike. Department of Child Development, Faculty of Medical and Pharmaceutical Sciences Kumamoto University Graduate School, Kumamoto, Japan; Department of Internal Medicine, Division of Hypertension, University of Texas Southwestern Medical Center, Dallas, TX; Division of Transgenic Technology Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan; Department of Oncogene Research, Research Institute 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, which is associated with a complex of proteins that spans the membrane and effectively links the cytoskeleton to the extracellular matrix. Despite the abundance of information about the molecular basis of this disease, there is currently no effective treatment for DMD because the mechanism by which dystrophin deficiency produces the clinical phenotype is poorly understood. Our previous data showed that dystrophin expression is normally detectable in smooth muscle cells (SMCs) as well as in skeletal muscle cells. Likewise, it is defective in SMCs of DMD patients and mdx mice, an animal model of DMD. Recent studies have revealed that focal multicellular myocytolytic lesions in muscular dystrophies may represent functional vascular abnormalities, regardless of whether or not they are primary. Although vascular dysfunction in DMD patients and mdx mice has been also reported, it is considered that the dystrophin deficiency of skeletal muscle and/or vascular endothelium, not vascular smooth muscle cells (VSMCs), is responsible for the vascular abnormalities.
We have hypothesized that dystrophin deficiency of VSMCs leads to vascular dysfunction and exacerbates muscle pathology. Here we generated transgenic mice expressing14Kb 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, that express dystrophin only in SMCs. The expression pattern was detectable by semi-quantitative RT-PCR analysis and immunohistochemical staining, which showed the specific expression of transgene in SMCs.
We assessed the vasoconstrictor response to sympathetic stimuli to determine if dystrophin expression of VSMCs affects the blood flow regulation during exercise. In contrast to mdx mice, alpha-adrenergic vasoconstriction in SMTg/mdx mice is significantly attenuated during muscle contraction, resulting in amelioration of the regulation of blood flow. Furthermore serum CK levels of SMTg/mdx mice were significantly reduced in comparison with those of mdx mice. This is the first report of a functional role for dystrophin in VSMCs and the resulting partial restoration of phenotype. These results suggest that VSMCs can be an effective target for treatment of DMD. Further, we believe that our SMTg/mdx mouse model is worth exploring to gain a better understanding of the functions of dystrophin in VSMCs and the pathophysiology of DMD patients.
 

10) An Ideal Therapeutic Agent for Duchenne Muscular Dystrophy Involving a Gutted Adenovirus Expressing Full-Length Utrophin

Jatinderpal R. Deol, Renald Gilbert, Mylene Bourget, Joon-Shik Moon, Josephine Nalbantoglu, George Karpati. Neuromuscular Research, Montreal Neurological Institute, Montreal, QC, Canada; Genomics and Gene Therapy Vectors, Biotechnology Research Institute, Montreal, QC, Canada; Department of Neurology, UMDNJ-New Jersey Medical School, Newark, NJ

Duchenne muscular dystrophy (DMD) is characterized by necrosis and progressive loss of muscle fibers. DMD patients have a mutation in the gene encoding dystrophin (dys), a large sarcolemmal membrane-associated cytoskeletal protein. Gene replacement therapy using fully deleted adenoviral vectors shows great potential for the eventual treatment of DMD.These vectors are less immunogenic than their predecessors and have the capacity to carry the full-length dys cDNA (13 kb). The introduction of fully deleted adenoviral (AdV) vectors encoding full-length dys into muscles leads to significant improvement of the dystrophic phenotype of the mdx mouse, an animal model of DMD. However, the introduction of dys, a neoantigen, into mdx muscle causes immune responses resulting in increased appearance of inflammatory infiltrates and damage to the muscle. An alternative approach is to use utrophin (utr), a functional homologue of dys, normally present only at the neuromuscular junction. This approach of simply increasing the expression of a protein that is already present in dystrophic muscle would prevent any immune response to the transgene product. Thus, we have created a fully deleted AdV encoding full-length murine utr cDNA regulated by a powerful hybrid promoter consisting of the chicken b-actin promoter and CMV enhancer. Cells infected with the purified recombinant AdV showed strong utr expression levels as determined by Western blot analysis. Furthermore, during in vivo studies, high transduction levels were obtained in the tibialis anterior (TA) muscles of mdx mice. The vector was administered to cohorts of neonates (2-4 day old) and adults (5 to 7 week old) at doses of 1.45 x 10¹⁰ and 4.35 x 10¹⁰ virus particles respectively. In the first group of neonates (n=5) at 10 days post-injection the mean number of sarcolemmal utr-positive fibers in injected vs control-injected TA was 1596 +/- 297 vs 114 +/- 76, which corresponds to 58 % transduction level. The mean number of utr-positive fibers in the adults (n=7) was 685 +/- 505 and 112 +/- 80 for viral and control injected TAs respectively, which corresponds to 23 % transduction level. The numbers obtained correspond to an approximate 14 and 6 fold increase in utr expression over control levels in neonates and adults. These results were further validated by consistent utr levels observed by Western blots using muscle sections. Subsequent in vivo evaluations will be performed at 30, 60, 90, 180, and 360 days post-injection. The injected muscles will be examined for utr expression, the restoration of the dys-associated protein complex, and the reversal of the physiological dystrophic phenotype. Considering our preliminary data thus far, helper-dependent, fully deleted (gutted) AdV expressing full length utr promises to be the ideal agent for gene replacement therapy in dys-deficiency states.
 

11) A Novel Approach of Gentamicin Therapy for Duchenne Muscular Dystrophy Using Hybrid Liposome and Establishment of a System To Identify the Patients Eligible for the Treatment

Shigemi Kimura, Kaori Ito, Shro Ozasa, Hiroe Kawano, Makoto Ikezawa, Makoto Matsukura, Ryuichi Ueoka, Yoko Matsumoto, Msafumi Matsuo, Yasuhiro Takeshima Teruhisa Miike. Department of Child Development, Kumamoto University Graduate School, Kumamoto, Japan; Department of Applied Life Science, Sojo University, Kumamoto, Japan; Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Aminoglycoside antibiotics have been found to suppress nonsense mutations located in the defective dystrophin gene in mdx mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD). However, the gentamicin therapy is hindered by several problems. First, a high dose of gentamicin, which is needed to induce a read-through of the nonsense mutations in DMD patients, can not be given because of oto- and nephrotoxicity. Even if this dose could be tolerated, this high concentration cannot be sustained due to the short half-life of gentamicin. Lastly, identifying patients that are applicable for this therapy is difficult because: 1) only 5 to 13% 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 kind of nonsense mutation.
In order to overcome the dose and concentration issues, we have investigated the use of hybrid liposomes. Mdx mice, given hybrid liposome encapsulated gentamicin (340mg/kg) intraperitoneally for 2 weeks, did not show oto- and nephrotoxicity. In contrast, only mdx mice injected with the same dose of gentamicin only exhibited ototoxicity. In addition, the efficiency of dystrophin positive fibers in the mdx mice injected with hybrid liposome encapsulated gentamicin was higher than in the gentamicin only injected mice; serum CK levels remained lower in the hybrid liposome group than in the gentamicin only group. Therefore, the results suggest that the hybrid liposome encapsulated gentamicin is more effective for DMD patients than genamicin alone. In order to resolve the patient identification problem, we have previously developed a system for identifying candidates that qualify for aminoglycoside therapy. Fibroblasts from 9 DMD patients with nonsense mutations of the dystrophin gene were isolated, induced to differentiate to myogenic lineage by AdMyoD, and exposed to gentamicin. The dystrophin expression in gentamicin exposed myotubes was monitored by in vitro dystrophin staining and western blotting analysis. The results showed that the gentamicin therapy is far more effective for DMD patients that have nonsense mutation TGA than for patients that have nonsense mutation TAA and TAG.
 

12) Efficient Expression of the 6 kb Mini-Dystrophin Gene by Trans-Splicing Adeno-Associated Viral (AAV) Vector Restores the Entire Dystrophin-Associated Glycoprotein Complex and Reduces Contraction-Induced Damage in the Mdx Mouse Model of Duchenne Muscular Dystrophy

Yi Lai, Yongping Yue, Mingju Liu, Arka Ghosh, John F. Engelhardt, Jeffrey S. Chamberlain, Dongsheng Duan. Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO; Department of Anatomay and Cell Biology, University of Iowa; Department of Neurology, University of Washington

Duchenne muscular dystrophy (DMD) is the most common lethal muscle disease caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) has been used to express the 3.8 kb C-terminal deleted micro-dystrophin gene for DMD gene therapy. Despite amelioration of some disease-associated changes, the microgene is not fully functional. Trans-splicing AAV vectors have been developed recently to express the 6 kb H2-R19 mini-dystrophin gene, the most competent gene besides the full-length gene. Our recent studies suggest that the RNA processing represents a critical barrier in trans-splicing AAV vector. In this study, we first screened a series of endogenous exon/intron/exon junctions by the RNase protection assay (RPA). The 56/56/57, 60/60/61 and 63/63/64 junctions hold the highest theoretic splicing values and may represent the most favorable sites to split the dystrophin gene. We generated synthetic mini-constructs carrying the splicing signals from each of above junctions. We also inserted the inverted terminal repeat junction in the middle of the intron. As a control, we included the 53/53/54 junction that has a low splicing value. The most efficient splicing was achieved in the 63/63/64 junction where the ratio of the spliced to unspliced RNA (S/U ratio) reached 7.1 0.9. The 60/60/61 and the 53/53/54 junctions yielded medium S/U ratios of 3.0 0.4 and 2.5 0.3, respectively. The 56/56/57 junction resulted in the lowest S/U ratio of 1.0 0.6. We also quantified the level of accumulated mRNA. Surprisingly, the highest mRNA was obtained from the 60/60/61 junction (255.1 20.6, relative unit), followed by the 63/63/64 (139.5 6.4), the 53/53/54 (48.8 20.6) and the 56/56/57 (0.8 0.2) junctions. We next generated two sets of AAV-6 trans-splicing vectors based on the 60/60/61 and the 63/63/64 junctions, respectively. 2 x 10¹⁰ particles of each set of vectors were delivered to the anterior tibialis (TA) muscle of 2-m-old mdx mice. Mini-dystrophin expression was quantified at 1 m post-infection by imunofluorescence staining. Consistent with the RPA results, trans-splicing vectors based on the 60/60/61 junction yielded 34 fold higher expression. To further evaluate the therapeutic potential, we delivered the 60/60/61 trans-splicing vectors (donor/acceptor co-infection, or donor or acceptor single infection) to both the TA and the extensor digitorium longus (EDL) muscles of the 2-m-old mdx mice. At 3 m post-infection, transduction efficiency in co-infected TA muscle reached 60-80%. Importantly, the entire dystrophin-associated glycoprotein complex (including dystroglycan, sarcoglycan, dystrobrevin and syntrophin) was restored in co-infected muscle. Furthermore, trans-splicing vector mediated mini-dystrophin expression provided significant protection against eccentric contraction induced injury in the EDL muscle.
 

13) Recombinant Adeno-Associated Viral (rAAV) Microdystrophin Vectors as Therapeutic Tools for Duchenne Muscular Dystrophy (DMD)

Takis Athanasopoulos, Ian Graham, Helen Foster, Norma Perez, Adelin Vulin, Vanessa Hill, Stewart Fabb, Luis Garcia, Olivier Danos, George Dickson. 1Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, United Kingdom; Gnthon III, Evry, Paris, Cedex, France

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder affecting 1:3500 male individuals, caused by recessive mutations in the dystrophin gene. The size of the gene (2.4Mb) and mRNA (14kb) in addition to immunogenicity problems and inefficient transduction of mature myofibres by currently available vector systems (that could incorporate the full dystrophin cDNA cassette) are formidable obstacles to the development of gene therapy. AAV vectors overcome many of the problems associated with other vector systems but accommodate limited transgene capacity (<5kb). More than 8 AAV vector serotypes have been identified to date with certain serotypes (1, 5, 6, 7) displaying more favourable tropism in transducing muscle fibers compared to the traditionally used AAV2.
A human microdystrophin cDNA (<3.8 kb) rationally designed in our lab based on patient and preclinical data incorporated and successfully packaged in an AAV-2 based vector cassette, under the control of a CMV promoter, effectively restored DAP complex and inhibited myofibre degeneration after local intramuscular administrations in nude/mdx mice, as reported previously. Other human microdystrophin versions have been developed in various labs worldwide displaying different degrees of functionality.
However certain issues including optimization of transduction, safety and immunogenicity vector/transgene profiling and species and tissue (muscle) specificity still remain to be fully addressed. Here we report data on production of human, canine and murine microdystrophin cDNA constructs via PCR-mediated overlap extension synthesis, incorporating either constitutive or muscle-specific promoter elements and optimal Kozak sequences for transcriptional improvement and packaging of the cassettes into recombinant AAV vectors of alternative serotypes (1, 5, 6) by pseudotyping. These vectors are currently fully screened and their functionality determined/compared by extensive in vitro and in vivo studies utilising local and systemic routes of administration (i.m., i.p., i.v. and intra-arterial) in dystrophic and non-dystrophic preclinical models.
 

14) Evaluation of Immune Responses to Dystrophin in Humanized mdx HLA-A*0201 Dystrophic Mice: Application in Duchenne Muscular Dystrophy Gene Therapy after U7 snRNA-Mediated Exon Skipping

Florent Ginhoux, Sabrina Turbant, Marylène Leboeuf, David A. Gross, François Lemonnier, Luis Garcia, Olivier Danos, Jean Davoust. Immunology, Genethon, Evry, France; Immunology, Institut Pasteur, Paris, France

Cellular immune responses may compromise long-term expression in Dystrophin-based gene therapy treatments. To predict cytotoxic T-cell responses mediated by dystrophin (DYST), we designed a new H-2-negative HLA-A*0201 transgenic mouse model breaded on mdx background bearing a stop mutation in DYST exon 23. After DYST plasmid injection we identified a shared mouse/human specific DYST epitope located on exon 24, which elicits HLA-A*0201-restricted cytotoxic T cell activities in all mice. We found however, that rescue of dystrophic muscle by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA (Goyenvalle A, Vulin A, Fougerousse F, Leturcq F, Kaplan JC, Garcia L, Danos O., Science. 2004, 306 : 1796-9) restores long term expression of DYST in muscle fibres and does not induce CTL activity against DYST in mdx HLA-A*0201 mice. This persistent exon skipping removes exon 23 and restores DYST exon 24 on the dystrophin messenger mRNA of the mdx HLA-A*0201 mouse. Several procedures were then attempted to induce muscle inflammation secondary to exon skipping. They failed to trigger effector CTL responses and failed to trigger immune rejection of rescued muscles fibres. We conclude that in contrast to delivery of DYST plasmid, rescuing dystrophic muscle through U7 snRNA-mediated exon skipping does not induce a primary immune response directed by exon 24 DYST sequence. The knowledge of HLA-A*0201-restricted human DYST peptides will be of importance to assay the occurrence of immune responses in HLA-A*0201 positive humans enrolled in future clinical studies.
 

15) Successful Myoblast Transplantation to Duchenne Muscular Dystrophy Patients

Jacques P. Tremblay, Daniel Skuk, Sylvain Michel, Raynald Roy, Jean-Guy Lachance, Hélène Senay, Deschênes Louise, Goulet Marlyne, Brigitte Roy, Jean-Pierre Bouchard. Gntique Humaine, Centre de Recherche du CHUL, Qubec, QC, Canada; Nphrologie, CHUQ Pavillon Hotel Dieu, Qubec, QC, Canada; Neurology, Centre Hospitalier Affili, Qubec, QC, Canada

Nine Duchenne muscular dystrophy (DMD) patients received injections of myoblasts obtained from skeletal muscle biopsies of normal donors. Cells were injected in 1 cm³ of the Tibialis anterior by 25 or 100 parallel injections. We performed similar patterns of saline injections in the contralateral muscles as controls. The patients received tacrolimus for immunosuppression. Muscle biopsies were performed at the injected sites 4 weeks later. We observed dystrophin-positive myofibers in the cell-grafted sites: in 8 of of 9 patients. In these 8 patients, the percentages of dystrophin positive fibers ranged from 3.5 to 26%. Eight patients had identified dystrophin-gene deletions and thus for four of these patients these results were obtained using mAbs specifically to epitopes coded by the deleted exons. Donor-dystrophin was absent in the control sites. For the other patients there were several folds more dystrophin-positive myofibers in the cell-grafted muscle than in the control muscle. Donor-dystrophin transcripts were detected by RT-PCR (using one primer reacting with a sequence in the deleted exons) in 8 patients. No antibodies were detected in the host serum against the donor myoblasts. Therefore, significant dystrophin expression can be obtained in the skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression.
 

16) Highly Efficient Exon-Skipping and Sustained Correction of Muscular Dystrophy Using an Adeno-Associated Viral Vector

Aurelie Goyenvalle, Adeline Vulin, Jean Claude Kaplan, France Leturcq, Olivier Danos, Luis Garcia. DMD Laboratory, Genethon & CNRS UMR8115, Evry, France; Laboratoire de Biochimie et de Gntique Molculaire, Hopital Cochin, Paris, France

In Duchenne Muscular Dystrophy (DMD), the lack of Dystrophin results in a series of catastrophic events that lead to muscle fiber death and muscle wasting. Dystrophin is a modular protein with a central rod domain composed of 24 spectrin-like repeats and truncated proteins missing some of these repeats can be fully functional or at least partly deficient as seen in patients with mild (Becker) forms of DMD. In a majority of severe DMD patients that presents out of frame alterations of the dystrophin transcript, a translational open reading frame would be restored by eliminating selected exon(s) from the pre mRNA. Exon skipping naturally occurs during dystrophin mRNA processing giving rise to rare revertant fibers that contain shortened proteins. It can be forced using antisense sequences delivered as synthetic oligonucleotides, or attached to a small nuclear (sn) RNA, notably U7. A double-target U7SmOPT containing antisense sequences from introns 22 and 23 (U7-SD23/BP22 ) was introduced in an AAV vector and packaged as an AAV2/1 pseudotype. Adult mdx mice were injected into the Tibialis Anterior, or submitted to whole limb arterial perfusion, and analysed between 2 and 13 weeks. Dystrophin transcripts missing exon 23 were detected by RT-PCR, representing 15% of the amplified material at two weeks and becoming the major species (60 to 70%) at one month. The Dystrophin protein was readily detected both by Western blot on muscle extracts and by immunofluorescence on tissue sections were restoration of the Dystrophin Associated Complex was also documented. The levels of rescued dystrophin was 3 % of normal at 2 weeks and 50 to 80% thereafter. The treated muscles were shown to recover normal contractile and mechanical properties by measuring resistance to eccentric contractions. Fiber resistance to exercise-induced damage was also restored, as seen using Evans Blue exclusion. The level and stability of U7-induced exon skipping that we observe in vivo in the context of an AAV vector is unparalleled, and provides a strong rational for the development of this approach in the clinic.
 

17) Lentivirus Mediated Dystrophin Expression in mdx Muscles

En Kimura, Sheng Li, Brent Fall, Miki Haraguchi, Leonard Meuse, Jeffery S. Chamberlain. Department of Neurology, University of Washington School of Medicine, Seattle, WA; Medicine and Biochemistry, University of Washington School of Medicine, Seattle, WA; Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, Seattle, WA

In dystrophic muscles of the mdx mouse, a model for Duchenne muscular dystrophy, activated-satellite cells (myoblasts) proliferate in the muscle micro- environment during regeneration and degeneration processes. Most of these cells terminally differentiate and form myofibers, while small numbers are stored adjacent to myofibers as mitotically quiescent satellite cells for future muscle regeneration.
Satellite cells and other stem cells are an attractive target for genetic modification by lentiviral vectors, since these vectors enable stable gene expression of a transgene following integration into the host cells genomic DNA. We have generated a series of lentiviral vectors that express various reporter genes and truncated mini- and micro- version of the dystrophin gene, and have tested their transduction ability in various myogenic precursors both in vitro and in vivo. Skeletal muscles of newborn mice may be considered a good target for lentiviral vectors, because of the relatively high numbers of activated muscle progenitor cells contributing to muscle growth and satellite cell pools. Therefore, we tried targeting satellite cells or muscle progenitor cells using lentiviral vectors expressing dystrophin micro-genes using several different approaches.
Much higher levels of transduction were obtained following intramuscular injection into neonatal muscles, which are rich with active myoblasts, compared with muscles from young and adult mice; both muscle fibers and primary cultured satellite cells stably expressed a GFP marker gene delivered via lentiviral vectors. Injection of lentiviral vectors expressing a functional micro-dystrophin/eGFP fusion protein into neonatal mdx muscles resulted in stable expression of dystrophin for at least one year, the longest time point current analyzed. Functional studies of the effects of this transduction are in progress. These data show that myogenic stem cells can be stably transduced with lentiviral vectors and can contribute to stable muscle regeneration in dystrophic muscle by enabling continuous expression of micro-dystrophin, which may have implications for gene therapy of Duchenne muscular dystrophy.
 

18) Plasmid-Mediated Gene Transfer in mdx Mice Using Mini- and Micro-Dystrophin Constructs

Leland E. Lim, Carmen Bertoni, Thomas A. Rando. Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA; Geriatrics Research, Education, and Clinical Center, VA Palo Alto Health Care System, Palo Alto, CA

The mdx mouse is a homologue of Duchenne muscular dystrophy (DMD), which is due to mutations in dystrophin, a subsarcolemmal protein associated with the actin cytoskeleton. Though the pathological changes observed in the mdx mouse are different from those of human DMD, the mdx mouse is an excellent model in which to study potential gene therapy approaches. One technique is the use of naked plasmid DNA as a method of gene transfer. Previous work has shown that dystrophin can be restored to the sarcolemma by intramuscular injection of a full-length dystrophin cDNA construct. However, expression has been limited to a small percentage of fibers close to the injection site. Furthermore, the large size of the dystrophin cDNA presents obstacles to the design of effective vectors and efficient uptake into muscle fibers. Here, we report the use of two truncated dystrophin plasmid constructs introduced using electroporation into the tibialis anterior muscles of young mdx mice. These constructs have large deletions of the central rod domain while preserving the critical N- and C-terminal regions. We tested whether these truncated constructs would be more efficacious than the full-length dystrophin cDNA by virtue of improved delivery and uptake. In order to follow the expression of dystrophin over time in living mice, we used a luciferase reporter construct introduced into muscle along with the dystrophin constructs. Luciferase activity can be followed non-invasively using a bioluminescent imaging system, and the persistence of luciferase activity in mdx mouse muscle is dependent upon the expression of dystrophin to prevent muscle fiber breakdown and concomitant loss of luciferase plasmids. Our results show that the truncated dystrophin constructs are at least as effective as full-length dystrophin in preserving luciferase activity. Also, sarcoglycan and dystroglycan components are present at the sarcolemma of the dystrophin-expressing fibers, indicating that the truncated dystrophin constructs are capable of restoring a functional dystrophin-glycoprotein complex. Current analyses are focusing on the relative distribution of plasmids in muscle to compare delivery of constructs of different sizes. We are also analyzing the effectiveness of dystrophin expression by assessing central nucleation. Finally, our current studies suggest that the longitudinal distribution along the length of individual fibers is critical to its ability to protect that fiber over the long term from the degenerative process. At this stage, we conclude that the smaller dystrophin proteins are effective in ameliorating the dystrophic features of mdx muscle fibers, and may be superior to full length dystrophin constructs for the purpose of plasmid-mediated gene transfer for the treatment of Duchenne muscular dystrophy.
 

19) Canine Mini-Dystrophin Gene Transfer by AAV1 in mdx Mice Ameliorates Dystrophic Pathology and Protects Membrane Integrity

Bing Wang, Juan Li, Liqiao Zhou, Chunping Qiao, Mengnan Tian, Tong Zhu, Janet Bogan, Joseph Kornegay, Xiao Xiao. Dept. of Molecular Genetics and Biochemistry and Dept. of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA; Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, MO

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorders, affecting one in 3,500 males. No efficacious treatment is currently available for DMD. While the mdx mouse is the most widely used small animal model for DMD, the large animal model, golden retriever muscular dystrophy (GRMD) dog, displays more similarities to human DMD patients in clinical and pathological characteristics. Therefore, the GRMD dog is considered a clinically more relevant and therapeutically more challenging DMD model.
To investigate AAV-mediated gene therapy for DMD in the canine model, we have cloned and constructed a canine version of mini-dystrophin gene. The dystrophin cDNA was partially cloned via RT-PCR from normal dog muscle. The N-terminus, 5 central rod domains and the cysteine-rich domain were spliced together, generating the canine mini-dystrophin gene of 3.8 kb, which was subsequently cloned into the AAV vector driven by the CMV promoter. Before testing the mini-dystrophin in dogs that have limited availability, we first tested the construct in the mdx mice to see if the primary biological functions could be observed.
We show that the canine mini-dystrophin gene is efficiently expressed in both neonatal and adult mdx mice at one to three months after AAV1 vector injection. Immunostaining of serial muscle thin-sections, using antibodies against rods 1 2 region of the dystrophin and against a, b, and g sarcoglycans separately, revealed the expression of canine minidystrophin gene and the restoration of the missing sarcoglycans at the plasma membrane. Importantly, the missing nNOS associated with myofiber membrane was also partially restored and coincided mini-dystrophin. H E staining of the vector-treated muscle displayed normal histology and the lack of fibrosis and infiltration, when compared to the contralateral untreated gastrocnemius. We found that the central nuclei have been reduced from 70% to 25% in adult mdx group, and over 98% of nuclei were peripherally located in areas of young mdx muscle expressing canine mini-dystrophin. Finally, in vivo muscle cell membrane integrity test after Evans blue injection also showed improvement and the exclusion of EBD dye leakage in mini-dystrophin-positive areas. Thus, the results obtained in mdx mice pave the way for further testing the canine mini-dystrophin in the GRMD dog model.
 

20) An AAV Vector-Mediated Gene Transfer into Canine Skeletal Muscle

Madoka Ikemoto, Katsutoshi Yuasa, Madoka Yoshimura, Akiyo Nishiyama, Yuko Miyagoe-Suzuki, John McC Howell, Shin'ichi Takeda. Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; Faculty of Pharmacy, Musashino University, Nishi-tokyo, Tokyo, Japan; Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, Western Australia, Australia

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene (14 kb cDNA). An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, but it has a limitation in insertion size up to 4.9 kb. We recently demonstrated that the AAV vector-mediated micro-dystrophin cDNA transfer could ameliorate dystrophic phenotypes in skeletal muscles of dystrophin-deficient mdx mice (Mol Ther 10: 821-828, 2004). For clinical application, it is important to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp Anim. 52: 93-97, 2003). To investigate transduction efficiency in canine skeletal muscle using an AAV vector, we injected the AAV vector encoding the LacZ gene driven by a CMV promoter (1.0-2.0 x 10¹³ vg/ml, 100-500 l/muscle) into skeletal muscles of normal dogs. -galactosidase (-gal) was expressed only in few fibers at 2 weeks after the injection, and not detected at 4 or 8 weeks after the injection. Instead, large numbers of mononuclear cells appeared around -gal-expressing fibers. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of the AAV vector, we examined viral infectivity in vitro, cytotoxicity and immune responses of AAV vector transduction in vivo. First, we infected the AAV vector into canine primary myotubes. This in vitro study showed that the AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particles elicit cytotoxicity or not. When a promoter-less AAV vector expressing no transgene (5 x 10¹² vg/muscle) was injected into canine muscle, almost no infiltrating cells was observed in injected muscle. Third, we investigated immune responses. A lot of CD4- or CD8-positive cells were detected in clusters of infiltrating cells, together with elevated serum level of anti--gal IgG. To confirm low transduction depending on immune response, dogs received daily oral administration of cyclosporine (20 mg/kg/day) from 5 day of the introduction of the AAV vector. Immunosuppression largely but not completely improves transduction efficiency of the AAV vector. These results suggest that AAV vector-mediated gene transfer elicits stronger immune responses in canine muscle, but immune responses against transgene products can not thoroughly explain the phenomenon. Cellular toxicity of transgene products might also participate in these infiltrations, while cytotoxicity and immunity of the AAV particles themselves can be negligible based on the result of a promoter-less AAV vectors. It is indispensable to know the molecular background of excess immune responses and cellular toxicity in canine models to establish AAV vector-mediated gene transfer in dystrophic patients.
 

21) Overlapping Adeno-Associated Viral (AAV) Vector Mediated Gene Transfer Is Dependent on Viral Serotype and the Transgene Sequence in Skeletal Muscle

Arkasubhra Ghosh, Yongping Yue, Dongsheng Duan. Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO

The small packaging capacity is one of the major hurdles for adeno-associated virus (AAV)-mediated gene therapy. The overlapping approach has been developed recently to expand the AAV packaging capacity (Duan et al, Mol. Ther. 4:383, 2001; Halbert et al, Nat. Biotechnol.20:697, 2002). In this approach, a large gene is split into two partially overlapping fragments and separately packaged into two AAV vectors, including the upstream (carrying the 5-end of the gene) and the downstream (carrying the 3-end of the gene) vectors. Transgene expression is achieved after homologous recombination of the overlapping region between the upstream and the downstream vectors in co-infected cells. Despite the promising proof-of-principle results in the lung with AAV-6 alkaline phosphatase (AP) overlapping vectors, the transduction efficiency in skeletal muscle has been very disappointing. With AAV-2 LacZ overlapping vectors, the efficiency is only 0.37% of that from an intact AAV-2 LacZ vector. This level of expression is far from sufficient to treat muscular dystrophy. In this study, we examined two potential rate-limiting factors in the overlapping approach, including AAV serotype and the transgene sequence. Previous studies suggest that AAV transduction in muscle is influenced by viral serotype. AAV-6 mediates much higher gene expression than AAV-2 in muscle. To test whether AAV-6 can improve overlapping vector-mediated gene transfer in muscle, we delivered a total of 1 x 10e9 viral genome particles of LacZ overlapping vectors (AAV-2 or AAV-6; 5 x 10e8 particles of each vector including the upstream and the downstream vectors) to the anterior tibialis (TA) muscle of 6-week-old BL10 mice. As a control, we also delivered 5 x 10e8 particles of the intact AAV LacZ vector to the contra-lateral TA muscle. Transduction efficiency was quantified at 6 weeks later by scoring the percentage of LacZ positive myofibers. Consistent with previous reports, less than 0.03% of myofibers were transduced by AAV-2 overlapping vectors. However, AAV-6 overlapping vectors yielded 35.2 5.7% transduction. This equals 42.25% of that of the transduction efficiency from an intact AAV-6 LacZ vector. To determine whether the transgene sequence effects the transduction efficiency, we compared AAV-6 LacZ and AAV-6 AP overlapping vectors. Surprisingly, the transduction efficiency of AP overlapping vectors (80.4 2.8%) reached that of the intact AP vector (84.1 1.9%). In summary, our findings suggest that AAV-6 overlapping vectors represent a promising approach to deliver certain larger therapeutic genes for muscular dystrophy gene therapy.
 

22) Evaluation of Gene Transfer Efficacy Mediated by AAV1 and AAV6 Vectors in Skeletal Muscle of Adult Mice Following Different Routes of Administration

Christel Rivière, Karine Poulard, Gabor Veres, Olivier Danos, Anne M. Douar. Gene Transfert Department - CNRS UMR 8115, Genethon, Evry, France

Skeletal muscle represents an important target tissue for gene therapy application due to the large number of genetic muscular disorders including Duchene Muscular Dystrophy. In addition the muscle may be also used as a delivery platform to express therapeutic proteins into the bloodstream for non-muscular metabolic disorders. Adeno-Associated Virus 1-based vector (rAAV1) is often considered as the most efficient AAV serotype to deliver genes into the muscles. In this study, we compared the performance of AAV pseudotyped (based on AAV2 genome) vectors expressing a secreted form of the murine alkaline phosphatase, (mSEAP) with capsid from serotype 1 and 6, in mouse skeletal muscle using different delivery methods: direct injection into individual muscle (intramuscular, IM), local limb distribution of vectors via the circulation (intra-arterial delivery, IA) or systemic delivery (intravenous administration, IV). We injected the maximal volume that each of these route allowed. We analyzed two parameters: the levels of a reporter circulating protein (), a relevant parameter for a depot organ strategy, and the extent of fiber transduction in the muscles, a relevant parameter in view of a muscular disorder therapeutic approach.
We compared rAAV1 and rAAV6 encoding mSEAP in a dose response study following IM administration in adult C57Bl/6 mice. Both vectors showed strong and equivalent levels of transduction at the highest doses (6x10⁸ vg and 3x10⁹ vg), while at the lowest dose injected (1.5x10¹⁰ vg), rAAV6 was 3-fold more efficient than rAAV1 in transducing muscle, suggesting a differential threshold of efficiency at sub-optimal doses.
In addition, the potential for systemic gene transfer after IV of rAAV1 and rAAV6 at the whole body level was investigated. For the two doses tested (1x10¹¹ vg and 3x10¹¹ vg). AAV6 vector led to 3-fold higher levels of circulating mSEAP levels than rAAV1. In addition, a widespread transduction of both skeletal and cardiac tissues was observed with rAAV6 after histochemical detection of mSEAP. However, in term of circulating protein level, IV administration was less efficient than IM injection since the same level of circulating mSEAP was achieved with 3x10¹¹ vg using IV delivery compared to3x10⁹ vg for IM delivery.
We have also evaluated intra-arterial delivery via the femoral artery of rAAV1 and rAAV6 at two doses (1x10¹¹ vg and 3x10¹¹ vg). This route yielded a 10 fold higher mSEAP levels in the serum than IV and led to robust transgene expression pattern in the hind limb muscles.
Finally, in an attempt to increase gene transfer efficacy in muscle after IV delivery, we co-injected the VEGF, with either rAAV1 or rAAV6. For both vectors, the presence of VEGF did not show a positive impact on transgene expression levels.
In conclusion, our results show that despite the high similarity between AAV1 and AAV6 capsid sequences, rAAV6 performance for muscle transduction are superior to rAAV1 with all IM, IA or IV delivery routes. This finding prompts us to consider AAV6 vectors as one of the most efficient viral agent to deliver gene in skeletal muscle and further clinical applications.
 

23) Identification of Differentially Expressed Genes in Duchenne Muscular Dystropy Utilising RNAi Technology: Possible Targets for Gene Therapy

Mohammad Mahdi Ghahramani Seno, Ian R. Graham, Ken Laing, Marita Pohlschmidt, Takis Athanasopoulos, Mark Crompton, George J. Dickson. Centre for Biomedical Research, Royal Holloway-University of London, Egham, Surrey, United Kingdom; Bacterial Microarray Group London, St Georges Hospital Medical School University of London, London, United Kingdom

Duchenne Muscular Dystrophy (DMD) is one of a group of genetically heterogeneous muscular dystrophies that are characterized by progressive weakness and wasting of skeletal muscle. Loss of myofibres occurs in response to a deficiency of dystrophin, a protein which is believed to be responsible for myofibre maintenance and integrity. Dystrophin forms a link between the cytoskeleton and the membrane-spanning dystrophin-associated glycoprotein complex (DAPC), indicative of a structural role for dystrophin.
The application of gene therapy protocols for DMD still presents many daunting challenges due partly to intrinsic features of the dystrophin gene. Hence, improvement in the understanding of the underlying primary molecular events leading to a dystrophic pathology might pave the way for the discovery of new starting points.
Here we present a strategy to use RNAi technology to study the events occurring in muscle cell development due to dystrophin deficiency. RNAi has been proven to be a powerful technology to study molecular effects due to knockdown of single genes. We have used a series of siRNAs to target and knock down the expression of dystrophin in primary cultures of mouse muscle, and subsequently used transcriptomic array analysis to identify genes whose expression was affected in response to dystrophin deficiency. The data obtained from this experiment, which include some very interesting potential new targets, are currently being analysed. We are also developing a recombinant adeno-associated virus (rAAV) vector expressing an shRNA targeting dystrophin. The use of such rAAV-shDNA vectors will enable us to target dystrophin in vivo to obtain a better and potentially curative insight into the pathophysiology of DMD.
 

24) Perivascular CD45^-:Sca-1⁺:CD34^- Cells Are Derived from Bone Marrow Cells and Participate in Dystrophic Skeletal Muscle Regeneration

Sheng Li, Morayma Reyes, En Kimura, Jessica Foraker, Miki Hagakura, Leonard Meuse, Brent Fall, Jeffrey S. Chamberlain. Department of Neurology, University of Washington School of Medicine, Seattle, WA; Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, Seattle, WA

Multiple mechanisms may account for bone marrow (BM) cell incorporation into myofibers following muscle damage. Here, we demonstrated that mouse CD45^-:Sca-1⁺:CD34^- cells may play a role in the maturation of skeletal muscles and regeneration of mdx^4cv dystrophic skeletal muscles, an animal model for Duchenne muscular dystrophy. To understand the origin of CD45^-:Sca-1⁺:CD34^- cells in mouse skeletal muscle, we reconstituted lethally irradiated wild type or mdx^4cv mice with unfractionated BM cells from transgenic mice ubiquitously expressing green fluorescence protein (GFP). 1, 2, and 6 months post-transplantation, we analyzed the skeletal muscle mononuclear cells from the recipients by flow cytometry for GFP, CD45-PerCP, Sca-1-PE, and CD34-APC. To our surprise, we found BM-derived (GFP⁺) CD45^-:Sca-1⁺:CD34^- cells in the skeletal muscles of these GFP⁺ BM transplant recipients. We also demonstrated that these BM-derived cells were localized in the perivascular tissue by immuno-staining and that their frequency increased with time. We were interested in the potential clinical application of these cells for muscle diseases. Thus, we sorted CD45^-:Sca-1⁺:CD34^- cells by fluorescence activated cell sorting (FACS) skeletal muscle mononuclear cells and cultured them in several stem cell media (recipes), including a low-serum medium containing specific cytokines for isolating multipotent adult progenitor cells (MAPCs). MAPCs can be isolated from skeletal muscle and BM and differentiate to form myotubes in vitro and in vivo. Strikingly, we found that MAPCs were enriched up to 40 folds by sorting this population from skeletal muscle mononuclear cells. Concomitantly with the increase in frequency of BM-derived muscle CD45^-:Sca-1⁺:CD34^- cells, frequency of BM-derived muscle MAPCs also increased with time post-transplantation in dystrophic muscles. Furthermore, BM-derived muscle MAPCs displayed similar phenotypes of endogenous muscle MAPCs, suggesting a potential mechanism of BM cell migration to dystrophic skeletal muscles. To understand how these BM-derived cells migrate to the muscle and once in the muscle how they mobilize, we investigated the in vitro chemotatic response of GFP⁺ (BM derived) muscle MAPCs and CD45^-:Sca-1⁺ cells isolated from muscles of GFP⁺ BM transplant recipients. We found that these cells were highly chemoattrated to stroma derived factor, SDF-1, a chemo-attractant for cells expressing CXCR4. We also observed higher frequency of BM-derived CD45^-:Sca-1⁺:CD34^- cells in mdx dystrophic muscle than wild type muscle, which may be explained by higher expression levels of SDF-1 in mdx dystrophic muscles. Taken together, our results suggest that dystrophic muscles recruit BM cells that localize in perivascular tissues and can be defined as CD45^-:Sca-1⁺:CD34^-. This population when cultured enriches for MAPCs and can participate in muscle regeneration in dystrophic muscles.
 

25) AAV-Mediated Myostatin Propeptide Gene Delivery Results in Growth and Hypertrophy of Skeletal but Not Cardiac Muscles

Chunping Qiao, Jianbin Li, Tong Zhu, Chunlian Chen, Terry O'Day, Jon Watchko, Juan Li, Xiao Xiao. Department of MGB, University of Pittsburgh, Pittsburgh, PA; Dept. of Pediatrics, University of Pittsburgh, Pittsburgh, PA; Dept. of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA

Muscular dystrophies are inherited myogenic disorders characterized by progressive muscle wasting and weakness. Because of the lack of effective treatment with traditional pharmaceutical agents, gene therapy and stem-cell therapy have been vigorously explored. Besides gene replacement therapy, a different form of gene therapy, i.e., expression of booster genes, has been investigated, aiming at alleviating the secondary deficiencies in muscular dystrophies rather than the primary ones. Blockade of myostatin is one of such strategies. Myostatin is a member of the TGF-beta family and a negative regulator of skeletal muscle growth. Studies using transgenic mice or antagonist antibody against myostatin showed promotion of muscle growth and amelioration of muscular dystrophy. However, gene delivery of myostatin inhibitors has not been reported.
In this study we examined whether myostatin propeptide gene transfer into normal mice by AAV vectors could increase muscle mass and strength. AAV vector carrying myostatin propeptide (MPRO) gene was delivered into neonate and adult BL10 mice respectively. For neonates study, AAV serotype 8 carrying MPRO gene was delivered into 3 to 5 days old mice by intraperitoneal injection. Two months after vector injection we observed very significant hypertrophy of the skeletal muscles. The treated mice gained body weight and had less fat when compared to the controls. The skeletal muscle mass including TA, GAS, Quadriceps, and Diaphragm from the treated mice were significantly larger (p<0.01). Myofiber diameters of the treated mice were larger than the untreated ones. The hypertrophy was not observed in the hearts of the treated mice despite the gene expression there. However, the larger muscle did not yield stronger force. The peak twitch and peak tetanic force of the treated muscle did not increase significantly, and the treated mice also did not increase performance on the treadmill. For the adult study, AAV-MPRO gene was delivered by local and systemically. Local intramuscular injection study indicated that the treated muscle mass also increased significantly. However, the treated muscle showed increased central nucleation, suggesting muscle regeneration. The study of systemically delivery of AAV-MPRO gene to the adult mice is under way. In conclusion, these data have shown for the first time that muscle mass can be increased by gene delivery of myostatin propeptide in normal mice. Further studies on muscular dystrophy animal models are warranted.
 

26) Real Time Imaging of Myoblast Transplantation Using the Human Sodium Iodide Symporter (hNIS) as Reporter Gene

Manaf Bouchentouf, Basma F. Benabdallah, Joel Rousseau, Marcel Dumont, Jacques P. Tremblay. Human Genetic unit, CHUL, Ste-Foy, QC, Canada; Radiology Unit, HSFA, Quebec, QC, Canada

BACKGROUND: The quantification of the graft success is a key element to evaluate the efficiency of cellular therapies for several pathologies such as Duchenne muscular dystrophy. This study describes a novel approach to evaluate the success of myoblast transplantation (i.e., the survival or the transplanted cells and of the muscle fibers that they have formed) by real-time imaging. METHODS: C₂C₁₂ myoblasts were first transfected with a plasmid containing the human sodium iodide symporter (hNIS) gene. The specific uptake of ^99mTCO₄ by the hNIS positive myoblasts was demonstrated in vitro, no accumulation of ^99mTCO₄ was observed within the control cells. The cells were then transplanted into the Tibialis anterior (TA) muscle of mdx mice. Following intra-peritoneal administration of Na^99mTCO₄, scintigraphies (g-radiographs) were performed depicting hNIS-dependent ^99mTCO₄ uptake within the TA. RESULTS: Preliminary experiments have demonstrated that the resolution of the camera used is 2 mm. In vitro the cells expressing the hNIS were able to incorporate the Na^99mTCO₄ and were also able to form myotubes. In vivo image acquisition of transplanted mice TA revealed that hNIS positive cells were able to incorporate the Na^99mTCO₄. CONCLUSIONS: This approach permitted to evaluate the progression of the transplantation and the graft success without having to biopsy the animals during the follow-up period.
 

27) Overexpression of the Myostatin Antagonist Follistatin in Normal Myoblasts Genetically Modified with a Lentivirus

Basma F. Benabdallah, Joel Rousseau, Manaf Bouchentouf, Jacques P. Tremblay. Human Genetic, CHUL-CHUQ-Laval University, Sainte Foy, QC, Canada

Background: Duchenne muscular dystrophy is a severe myopathy caused by the absence of a functional dystrophin in muscles. Transplantation of normal myoblasts is a potential therapy that permits to restore the expression of the dystrophin in transplanted muscles by the formation of hybrid dystrophin positive fibers. However, the success of this approach is compromised by the limited regeneration of damaged muscle. Myostatin, the most powerful inhibitor of muscle growth identified to date, is regulated by different antagonist proteins such as follistatin. Our purpose is to block the myostatin signal in mdx and SCID host mice by the respective transplantation of normal murine and human myoblasts genetically modified with a follistatin lentivirus.
Methods: 293T packaging cell line was used to produce a lentivirus coding for the short form of follistatin protein under the control of a cytomegalovirus promoter. Normal murine and human myoblasts were infected with the lentivirus to overproduce the follistatin protein. The surexpression of the follistatin in both transfected 293T cells and infected murine and human normal myoblasts was verified by immuno-cytochemistry assay and Western blot. A lentivirus containing the eGFP gene was used as a control. The differentiation of control and lentivirus-follistatin infected myoblasts was also evaluated by the determination of the fusion index.
Results: The immunocytochemistry assay showed that the follistatin was clearly overexpressed in both pCMV-Fst transfected 293T cells and infected murine and human normal myoblasts compared with the control cells transfected or infected with the eGFP lentivirus. The Western blot also demonstrated that the follistatin was effectively overexpressed in pCMV-Fst transfected 293T cells and infected murine and human normal myoblasts compared with the control cells transfected or infected with the eGFP lentivirus. The fusion index test also showed that myoblasts infected with the follistatin lentivirus formed more myotubes than myoblasts infected with the eGFP-lentivirus when cultured in differentiation conditions.
Conclusions: The transplantation of normal murine or human myoblasts overexpressing follistatin, in mdx or SCID mouse respectively in order to antagonize the myostatin signaling, could be a good approach to improve the success of the potential cellular therapy of Duchenne myopathy.
 

28) Development of AAV-Mediated Gene Therapy for Murine Models of Genetic Diseases Affecting the Heart

Christina A. Pacak, Cathryn Mah, Gabriel Gaidosh, Melissa Lewis, Raquel Torres, Kevin Campbell, Glenn A. Walter, Barry J. Byrne. Molecular Genetics and Microbiology, University of Florida, Gainesville, FL; Powell Gene Therapy Center, University of Florida, Gainesville, FL; Physiology, University of Florida, Gainesville, FL; Cellular and Molecular Therapy, University of Florida, Gainesville, FL; HHMI, University of Iowa, Iowa City, IA

The long term goal of this project is to develop a clinically relevant gene therapy approach for the treatment of genetic diseases affecting the heart. Due to its small size, safety and proven ability to persist for long periods of time in muscle, adeno-associated virus (AAV) has emerged as a promising cardiac gene delivery vehicle. We have sought to determine the most advantageous combination AAV serotype and vector delivery route for the transduction of cardiomyocytes in vivo. Both intra-venous (iv) and intra-cardiac (ic) injection routes were compared by injecting 1x10¹¹ and 5x10¹⁰ (respectively) vector particles of AAV-CMV-LacZ per mouse neonate of 3 different serotypes AAV1, AAV8 and AAV9. Tissue analysis included both x-gal staining on tissue sections to visualize expression and the quantitative -galactosidase enzyme detection assay. Our results show that iv administration of AAV9 results in 30-fold more efficient transduction of cardiac tissue than AAV1. Moreover, hearts injected with AAV9 displayed a global distribution of transgene expression suggesting this serotype has no transduction site preference within cardiac tissue. 2 murine models for cardiac dysfunction; the alpha-sarcoglycan (asg ^-/-) knockout model for Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) and the acid-alpha glucosidase (gaa^-/-) knockout model for Pompe Disease are currently being characterized functionally and morphologically using both non-invasive MRI and histological techniques. LGMD-2D is the result of mutations in the alpha sarcoglycan (ASG) gene. This model displays the development of dystrophic lesions in cardiac and skeletal muscle. We have developed a non-invasive assay using MRI that enables us to locate and measure the random development of lesions within the muscles of asg ^-/- mice. We have demonstrated functional correction and prevention of disease progression in skeletal muscles injected with AAV-ASG. Studies are currently underway to demonstrate the same effect in cardiac tissue. The cells of the gaa^-/- mouse contain lysosomes enlarged with glycogen due to a GAA enzyme deficiency. Previously, our lab has demonstrated that delivery of the human GAA gene to the skeletal muscle of this model is therapeutically beneficial. To evaluate gene delivery to the cardiac tissue of this model we are currently characterizing the cardiac phenotype of disease and assessing our ability to prevent disease presentation following optimized delivery of the human GAA gene. In conclusion, our maker-gene study has established that iv delivery using AAV9 is the most clinically advantageous combination of AAV serotype and delivery route for the transduction of cardiac tissue in vivo. We are now in the process of using this delivery method to demonstrate disease correction in two mouse models of cardiomyopathy.
 

29) Decorin Promotes Differentiation of Myoblasts into Myotubes That Express Slow MyHC In Vitro

Yong Li, Juan Li, Ying Tang, Xiao Xiao, Johnny Huard. Dept. of Orthopaedic Surgery, Childrens Hospital and University of Pittsburgh, Pittsburgh, PA; Dept. of Molecular Genetic and Biochemistry, University of Pittsburgh, Pittsburgh, PA

Decorin, a small leucine-rich proteoglycan, is a key regulator in extracellular matrix assembly and cell proliferation. It also has a specific effect on the migration and differentiation of embryonic skeletal muscle cells (1). We have demonstrated that decorin can prevent muscle fibrosis, enhance muscle regeneration, and improve the functional recovery of injured skeletal muscle (2). However, the mechanism behind decorins effect on muscle regeneration is unclear. In the current study, we used an AAV-mDecorin plasmid to transfect a mouse decorin gene into myoblasts (C2C12 cells) and evaluated the phenotypic changes during the resultant myoblast differentiation. Our results revealed that myoblasts engineered to express decorin undergo differentiation more readily than do normal myoblasts in vitro. This enhanced differentiation may be due to the stimulation of P21, an important cyclin-dependent kinase inhibitor that is up-regulated during muscle cell differentiation (3). We also observed up-regulated expression of myogenic proteins (MyoD, Myogenin, Myf5, and Myf6) by these decorin-transfected (CD) cells. This study demonstrates that decorin treatment resulted in the overexpression of slow myosin heavy chain (MyHC) and the production of slow myotubes by CD cells in vitro. These results suggest that decorin may promote muscle regeneration through the activity of slow MyHCexpressing muscle fibers. These results indicate that decorin improves skeletal muscle healing not only by preventing fibrosis, but also by promoting muscle regeneration. These findings could explain why decorin treatment can lead to nearly complete functional recovery of injured muscle (2).
References:
1. Villena J, et al. J Cell Physiol 2004;198:16978.
2. Fukushima K, et al. Am J Sports Med 2001;29:394402.
3. Halevy O, et al. Science. 1995;267:1018-21.