Abstract No. 1119

Comparison of a Series of Mini-Dystrophin Genes in Protection of Dystrophic Muscles in mdx Mice

Bing Wang,1 Juan Li,1 Xiao Xiao.1

1Dept. of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, where mutations in the dystrophin gene cause the absence or profound reduction of dystrophin protein. Dystrophin is an elongated protein and localized along the inner face of sarcolemma to protect cell membrane integrity. Previously, we showed that dystrophin gene can be substantially reduced to one third (1/3) of its full-length coding sequence without compromising essential functions. The mini-dystrophin molecules with large deletions in C terminus and rod domains (maintaining 5 ~ 6 rod repeats) can be delivered by AAV vector and act to prevent the development of muscular dystrophy in young and adult mdx mice.

In the report, we wished to define the minimal functional domains and the minimal length of the dystrophin protein required to protect the muscle. We generated a series of mini-dystrophin driven by MCK promoter in AAV vector, which include 1 to 5 rods and 2 hinges in the central rod domains. Our study showed that the mini-dystrophins maintaining at least four rod repeats (R1, R2, R22, and R24) and 2 hinges (H1 and H4) in the central rod domain retained essential functionality, while mini-dystrophin with five rods exhibited the best protective effects to the dystrophic muscle. However, mini-dystrophins containing 3 rods or less could not prevent the histopathological morphology and central nucleation of mdx mice. Our study demonstrated that AAV vectors carrying the mini-dystrophin genes (at least 5 rods) achieved efficient and stable correction of major biochemical and physiological defects in dystrophic muscle.


Abstract No. 5

Protein Trans-Splicing as a Novel Strategy for Duchenne Muscular Dystrophy Gene Therapy with AAV Vectors

Juan Li,1 Paul X. Q. Liu,2 Bing Wang,1 Xiao Xiao.1

1Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States;
2Biochemistry Department, Dalhousie University, Halifax, Nova Scotia, Canada

Duchenne Muscular dystrophy (DMD) is the most common form of X-linked genetic disease, with a worldwide incidence of one in 3500 male birth. There is yet no effective treatment for this devastating disease. In this study, we have used a novel strategy termed intein-mediated protein trans-splicing to let the AAV vectors produce large proteins, whose coding sequences are larger than the 5 kb of a single AAV vector packaging limit. An intein is a protein sequence embedded within a precursor protein and it can excise itself via protein splicing. Therefore, an intein in protein is similar to an intron in RNA. A split intein can catalyze the trans-splicing (ligation) of two proteins. The target protein we used here is the Becker-form dystrophin encoded by a 6.3-Kb cDNA. The 6.3-kb gene was split in the center. A pair of half-genes was generated and modified with a selected intein sequence. These two half genes were separately packaged by AAV vectors. In vitro study by co-infection into 293 cells showed that a full-length Becker-from dystrophin protein was produced by the mechanism of protein trans-splicing from the two half-proteins. In vivo study by co-injection of the two AAV vectors into dystrophic mouse muscle demonstrated efficient dystrophin synthesis and correct localization of the protein in the myofibers. Most importantly, the trans-spliced dystrophin protected muscle cell morphology and prevented pathology, such as central nucleation caused by muscle cell degeneration and regeneration. In conclusion, We have demonstrated for the first time that an intein, particularly a split-intein, could work in the mammalian cells, both in vitro and in vivo. The protein trans-splicing strategy worked efficiently to ligate the two separate pieces of dystrophin half-proteins together to generate a functional dystrophin. Our preliminary data also showed that the protein trans-splicing method is much more efficient in generating full-length Becker-form dystrophin than the split (dual) AAV vector method via DNA trans-splicing. Therefore, protein trans-splicing could potentially be used for gene therapy of Duchenne muscular dystrophy and other diseases involving large genes.

Abstract No. 1131

A Variant of Human Dihydrofolate Reductase Can Be Used To Select Myoblasts for Ex Vivo Gene Therapy of Duchenne Muscular Dystrophy

Philippe Campeau,1 Marlyne Goulet,1 Jacques P. Tremblay.1

1Human Genetics Research Unit, CHUL, Laval University, Ste-Foy, Qué
bec, Canada

Ex vivo gene therapy for Duchenne muscular dystrophy (DMD) by transplantation of genetically corrected myoblasts is a promising approach, but the corrected myoblasts should be selected with a nonimmunogenic and thus human gene. The human dihydrofolate reductase (DHFR) gene is a potential selection gene and we tested whether it could eventually be used to select myoblasts. A vesicular stomatitis virus G (VSV-G) envelope pseudotyped MoMLV retrovirus containing the enhanced green fluorescence protein (EGFP) cDNA, an internal ribosomal entry site element (IRES) and a L22Y mutant DHFR cDNA was produced. It was used to infect myoblasts from a DMD patient which were previously immortalized with SV40 large T antigen and hTERT. The cells were selected with methotrexate. The ability of these genetically modified and selected myoblasts to fuse in vitro and in vivo after transplantation in the Tibialis anterior muscle of Severe Combined Immunodeficient (SCID) mice was assessed. The infected human myoblasts were found able to fuse in vitro and the resulting myotubes expressed human spectrin, a marker of differention. The selected myoblasts were also able to fuse in vivo in SCID mice and produced muscle fibers expressing human spectrin. Human DHFR is thus an appropriate selection gene for ex vivo gene therapy of DMD, and could be used in future clinical trials of myoblast transplantation for other diseases.


Abstract No. 1129

High Efficiency Plasmid Gene Transfer into Dystrophic Muscle

Dominic J. Wells,1 Helen Gollins,1 Jill McMahon,1 Aurora Ferrer,1 Kim E. Wells.1


The use of in vivo electroporation has dramatically improved the efficiency of naked plasmid DNA based gene transfer into skeletal muscle. However the use of strong electrical fields is often associated with substantial muscle damage and whilst normal muscle recovers quickly, such damage is inappropriate for dystrophic muscle. We have previously demonstrated high efficiency plasmid gene transfer to normal mouse skeletal muscle can be achieved with minimal damage by treatment of the muscle with hyaluronidase prior to electrotransfer of plasmid DNA (McMahon et al., 2001). We have now extended this work to examine the efficiency of this process in dystrophic muscle of the mdx mouse, a model for Duchenne muscular dystrophy. Initially we analysed [BETA]-galactosidase reporter gene expression and demonstrated similar total expression and percentage of transfected fibres in normal and dystrophic mdx muscle despite the myopathy and fibrosis in the latter muscles. In neither case was there a reduced efficiency in old compared to young muscle. This is in marked contrast to the use of many viral vectors. Increasing the amount of hyaluronidase in the pre-treatment had no significant effect but was associated with increased pathology in the treated muscles. No difference was observed between male and female mdx mice. The number of dystrophin positive fibres following plasmid transfer using the optimised protocol was also increased. In the absence of a dystrophin coding sequence in the plasmid there was no change in the number of dystrophin positive (revertant) fibres associated with the pre-treatment and electrotransfer. When a mouse dystrophin cDNA was used expression was stable out to the last time point measured (8 weeks) but transfer of a similar plasmid carrying a human dystrophin cDNA lead to a rapid loss of transfected fibres. These results confirm our previously published observations following standard intramuscular injections of dystrophin plasmids (Ferrer et al., 2000). Long term expression of murine dystrophin was also maintained in the mdx3cv following electrotransfer of plasmid DNA despite this mutant failing to express the smaller isoforms of dystrophin. We conclude that it is the presence of the rare revertant fibres that provides tolerance to dystrophin gene transfer.


Ferrer, A., Wells, K. E. and D. J. Wells (2000) Immune responses to dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Gene Therapy 7:1439-1446.

McMahon, J.M., Signori, E., Wells, K.E., Fazio, V.M. and D.J. Wells (2001) Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase - increased expression with reduced muscle damage. Gene Therapy 8:1264-1270.


Abstract No. 1122

Generation of Functional Muscle in Mdx Mice by Stem Cell Transplantation

Zhuqing Qu-Petersen, John Mytinger, Bridget Deasy, Arvydas Usas, James Cummins, Johnny Huard.

1Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, United States

Extensive efforts have been made to develop strategies to alleviate muscle weakness in Duchenne muscular dystrophy (DMD) by utilizing cell transplantation and cell based gene therapy. Traditional cell transplantation by using myogenic progenitor, myoblast, is feasible, but greatly limited by the low survival rate, poor spread, and immunorejection of the donor cells. The development of stem cells for transplantation may overcome these limitations, since stem cells are capable of long-term proliferation, high self-renewal, and multilineage differentiation, all of which may improve the long-term survival of transplanted cells. We recently isolated a rare population of muscle-derived stem cells (mdsc) from skeletal muscle of normal mice. This population of cells expressed hematopoietic stem cell markers, and retain the phenotypes for more than 30 passages. The transplantation of mdsc into dystrophin deficient muscle of mdx mice (an animal model for DMD) regenerated 10 times more dystrophin(+) myofibers than transplantation of the same number of satellite cell derived myoblasts for more than 3 months without immunorejection. Further investigation by tracking the donor cells (LacZ expression) in peripheral nerve and blood vessels demonstrated that mdsc displayed the capacity of differentiating into nerve satellite cells (Schwann cells), and vascular endothelial cells in transplanted dystrophic muscles. The results suggest that the pluripotent mdsc, capable of restoring myofibers and other structures required for normal muscle tissue, may contribute to the survival of transplanted cells via the formation of functional muscle in dystrophic mice. This population of cells may potentially be used in both cell and cell-based gene therapy to alleviate the muscle weakness in DMD as well as for other muscular dystrophies, in which transplantation of myoblasts is beneficial but its success extremely limited.


Abstract No. 1133

Gene Transfer into Skeletal Muscles Mediated by Electric Pulses. Optimization of Transfer Efficiency in Mdx Mice

Olga V. Ostapenko,1 Alexander N. Baranov,1 Eugenia A. Lesina,1 Vladislav S. Baranov.1

1Lab. of Prenatal Diagnosis of Inherited Diseases, Ott Institute of Obstetrics and Gynecology, Saint-Petersburg, Russian Federation

Duchenne muscular dystrophy (DMD) is the most prevalent of all neuromuscular disorders caused by dystrophin gene mutations affecting one in 3500 male births. It has been reported that electroporation can facilitate naked DNA gene transfer into skeletal muscles. This technique enhances plasmid delivery and expression of reporter or therapeutic genes.[br]Expression efficiency of mini-dystrophin gene (pSG5dys) and marker gene lacZ ( pCMV-nls-lacZ) constructs delivered into skeletal muscles by electroporation is reported. Different conditions of electroporation such as voltage, duration of electric pulses, time interval after injection and electroporation were tried. Morphological changes in the skeletal muscles of mdx-mice - biological tools of DMD and in control C57/BL/10J+/+ mice after electrical pulses application were investigated. Co-transfection of skeletal muscles of mdx-mice with both pSG5dys and pCMVnlsLacZ genes after application of electric pulses 10V and 20 V resulted in 7,5 % and 8.2% dystrophin positive muscular fibers (DPMF) respectively without visible changes in myofiber structure and necrotic lesions . Higher field strength ( over 50V) induced significant damage of muscular fibers. Changes in duration of electroporation from 10 to 50 sec., at the voltage 20 V and pulse frequency 1000 Hz has resulted in elevation of DPMV number up to 9,7%. Both genetic constructions demonstrated stable 2 weeks expression with maximal DPMV score (10,5%) on day 10 after electroporation. The experiments on transfection efficiencies of electroporation respective of other electric -pulses conditions and in combination of electric pulses with non-viral gene delivery vectors are in progress. Feasible implication of electric pulses for Duchenne Muscle Dystrophy treatment are discussed.


Abstract No. 41

Progress toward Gene Therapy of Duchenne Muscular Dystrophy Using Truncated Four-Repeat Dystrophin and AAV6

Scott Q. Harper,1 Michael J. Blankinship,1 Dongsheng Duan,2 Hollie A. Harper,1 Robert W. Crawford,1 Christine L. Halbert,3 John F. Engelhardt,2 Dusty Miller,3 Jeffrey S. Chamberlain.1

1Department of Neurology, University of Washington School of Medicine, Seattle, WA, United States;
2Department of Anatomy and Cell Biology, University of Iowa School of Medicine, Iowa City, IA, United States;
3Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, United States

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by mutations in the dystrophin gene. Studies in the mdx mouse model for DMD have provided hope that dystrophin gene replacement may be an effective therapy for this disease. Still, gene therapy for DMD is complicated by the: (1) large size of the gene (14 kb cDNA) that precludes its packaging into most known viral vectors; (2) requirement to deliver functional dystrophin to a large mass of muscle cells; (3) necessity for long term gene expression that will likely require evasion of the host's immune system. Adeno-associated viral (AAV) vectors possess features that may help to achieve the latter two requirements, but are hampered by their small cloning capacity (<5 kb). We have recently characterized extremely small dystrophin transgenes encoding large rod domain truncations that can circumvent the small packaging size of AAV. Our "micro-dystrophins" are ~3.8 kb in size, contain only four of the 24 dystrophin spectrin-like repeats, and achieved a functional correction of DMD in transgenic mdx mice. Furthermore, an AAV2 viral vector carrying the best micro-dystrophin (ΔR4-R23) caused a dramatic reversal of the morphological features associated with dystrophy in mdx mice that had already shown a significant pathology. Despite this, the amount of muscle transduction achieved using AAV2 vectors may be insufficient to provide a significant clinical benefit to a human patient. Consequently, we compared gene expression levels in vivo using two different AAV serotypes, the more highly characterized AAV2, and AAV6. We found using βgal reporter assays that AAV6&cdot;CMV-βgal led to 230 times more βgal expression than AAV-2&cdot;CMV-βgal 12 days after infection of mdx mouse muscle. However, we measured a dramatic drop in βgal expression 24 days post-injection using AAV6, but not AAV2. Our data suggest that there may be a dose-dependent immune response against βgal. Previous reports have suggested that muscle-specific expression may help avoid the immunogenicity problems created by expression of foreign epitopes in dystrophic muscle. Our data suggest that muscle specific expression was not sufficient to prevent the loss of βgal activity in mdx mice over time. However, more importantly, we achieved complete saturation of a tibialis anterior (TA) muscle from a young adult mdx mouse using AAV6 micro-dystrophin driven by a highly active muscle specific promoter (CK6), and dystrophin expression appeared to increase with time post-injection. We are currently assessing the immune responses against both AAV6&cdot;CK6-βgal and AAV6&cdot;CK6-micro-dystrophin in mdx mice using chromium release assays. In addition, we are attempting to measure a physiological correction of DMD in mdx mice injected with AAV&cdot;micro-dystrophin. These studies will provide important data to help assess the feasibility of scaling up our system for potential human clinical trials.


Abstract No. 1117

Adeno-Associated Virus Vector-Mediated Gene Transfer into Dystrophin-Deficient Skeletal Muscles Evokes Enhanced Immune Responses

Katsutoshi Yuasa,1 Miki Sakamoto,1 Yuko Miyagoe-Suzuki,1 Aki Tanouchi,1 Madoka Yoshimura,1 Hiroshi Yamamoto,2 Jane Li,3 Jeffrey S. Chamberlain,4 Xiao Xiao,3 Shin'ichi Takeda.1

1Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan;
2Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan;
3Department of Molecular Genetics and Biochemistry, Gene Therapy Center, and Duchenne Muscular Dystrophy Research Center, University of Pittsburgh, Pittsburgh, PA, United States;
4Department of Neurology, University of Washington School of Medicine, Seattle, WA, United States

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disease caused by a mutation in the DMD gene, which encodes 14 kb dystrophin cDNA. An AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach for treatment of DMD, since the vector allows long-term expression of the transgene with less immune response. We are attempting an effective gene transfer using an AAV vector into dystrophin-deficient mdx skeletal muscle. When the AAV vector encoding the lacZ gene driven by a CMV promoter (AAV-CMVLacZ) was introduced, transduction efficiency was significantly lower in mdx muscle than in control C57BL/10 (B10) muscle and β-galactosidase (β-gal) expression markedly decreased in mdx muscle 4 weeks after injection. Large numbers of cellular infiltration, including CD4- or CD8-positive cells, were observed around β-gal-expressing fibers in the AAV vector-injected mdx muscle. IgG antibodies against β-gal were largely detected in sera and muscles of the AAV vector-injected mdx mice than in those of B10 mice, while IgG antibodies against AAV particle showed similar responses in both mice. Furthermore, persistent immune suppression with anti-CD4 antibodies (GK1.5) prolonged β-gal expression in mdx muscle at least until 4 weeks post-injection. These data demonstrated that immune responses against the transgene product were largely induced in mdx muscle after the transfer of AAV-CMVLacZ. To assess whether antigen-presenting cells (APCs) are involved in activation of immune responses against the transgene product in mdx mice, we injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle degeneration. The AAV vector administration evoked substantial immune responses even in CVBA3' muscle, although dendritic cells or macrophages were barely detected in native CVBA3' muscles. Immune responses could have been activated in mdx muscle mainly by neo-antigens leaking from the muscle fibers rather than by ubiquitous expression of the transduced gene in APCs. To elucidate this hypothesis, we injected the AAV vector, where the lacZ gene was driven by a muscle-specific MCK promoter, into mdx muscles. β-gal expression remarkably decreased after 8 weeks in mdx muscles, although the expression was maintained at least for 24 weeks in B10 muscles, again indicating that mdx muscle fibres themselves are causes of immune responses. Transfer of a therapeutic gene such as micro-dystrophin by AAV vector may reduce immune responses, since a therapeutic gene product may protect the dystrophic fibers from leaking process of neo-antigens.


Abstract No. 714

Dystrophin Delivery in the mdx Heart Based on Ex Vivo Gene Delivery Using Muscle Derived Stem Cells

Thomas R. Payne,1 Yiqun Ling,2 Hideki Oshima,2 Tetsuro Sakai,2 Ryan C. Murdoch,1 James H. Cummins,3 Yong Li,3 Zhuqing Qu-Petersen,3 Makato Ikezawa,3 Ryan J. Pruchnic,3 Ronald J. Jankowski,1 Johnny Huard.1,3

1Bioengineering, University of Pittsburgh, Pittsburgh, PA, United States;
2Surgery, University of Pittsburgh, Pittsburgh, PA, United States;
3Orthopaedic Surgery, Molecular Genetics and Biochemistry, Children's Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA, United States

Patients that suffer from diseases of the cytoskeleton, such as the muscular dystrophies, progressively develop cardiomyopathy. Duchenne muscular dystrophy (DMD) is an X-linked muscle disease caused by mutations in the dystrophin gene. The gene product, dystrophin, is an important linkage component connecting the subsarcolemmal cytoskeleton and the extracellular matrix. Consequently, cardiomyocytes lacking dystrophin have been shown to be more susceptible to develop sarcolemmal injury and contractile dysfunction when subjected to increased levels of biomechanical stress (Danialou et al 2001). In this study, we investigated the use of muscle-derived stem cells (MDSCs), a recently isolated and identified stem cell population, as a novel cell source for ex vivo gene therapy to deliver dystrophin into the heart of an animal model for DMD. MDSCs, isolated from skeletal muscle of normal 3-week-old mice, express stem cell markers and have been found capable of differentiating in various lineages. These cells were then transduced with a retroviral vector MFG-NB containing a modified LacZ reporter gene (nls-LacZ), which includes a nuclear-localization sequence cloned from the simian virus (SV40) large tumor antigen and is transcribed from the long terminal repeat (LTR) (van Deutekom et al 1998). Through a left thoracotomy, transduced cells (1-5 x 105) were injected with a 30-G syringe into the left ventricular free wall of SCID/mdx mice, a dystrophin and immune deficient animal model. At 2 weeks following transplantation, the hearts were cryopreserved and sectioned for histological and immunohistochemical studies. Our results show that the transplanted MDSCs survived and differentiated into LacZ and dystrophin positive myotubes and myofibers within the native myocardium. This study demonstrates the feasibility of ex vivo gene therapy based on MDSCs to the deliver therapeutic proteins, including dystrophin, in diseased myocardium. Further experiments will be performed to evaluate the differentiation capacity of the MDSCs within the myocardium.


Abstract No. 44

Muscle Stem Cells Differentiated into Hematopoietic Lineage While Preserving Their Myogenic Potential

Baohong Cao,1 Shigemi Kimura,2 Makoto Ikezawa,1,2 Ryan Pruchnic,1 Jeffrey G. Kuremsky,1 James Cummins,1 Johnny Huard.1

1Orthopaedic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, PA, United States;
2Child Development, Kumamoto University, Kumamoto, Japan

We have isolated a population of muscle derived stem cells (MDSC) that express stem cell markers and are capable of differentiating in different lineages. A MDSC clone, mc13, was isolated from dystrophic mdx mice (an animal model of Duchenne muscular dystrophy) and genetically engineered to express b-galactosidase, human mini-dystrophin and neomycin resistance genes. Intravenous dissemination of mc13 cells to lethally irradiated adult mdx mice increased the life span of these mice. Upon examination of the recipients[ssquote] bone marrow we observed donor derived cells expressing CD45(+), Gr-1(+), and Mac-1(+), which consisted of 22%, 12%, and 14% of the host bone marrow cells respectively. Recovered bone marrow from the first recipients was also capable of differentiating into hematopoietic lineage and increasing the life span of secondary lethally irradiated recipients. After further selection of recipient[ssquote]s bone marrow with G418, the donor cells were transplanted intramuscularly in mdx skeletal muscle. The transplanted cells were still found capable of differentiating into muscle, although their myogenic capability was decreased when compared to the original mc13 cells. Our study demonstrated that muscle stem cells that have already differentiated into hematopoietic lineage retain their myogenic potential (myogenic) when transplanted into skeletal muscle. These results further validate the plasticity of muscle stem cell and that the environment plays a major role on the cell differentiation.


Abstract No. 1127

Gutted Adenoviral Vector Delivery of Full-Length Dystrophin or Utrophin to mdx Mouse Skeletal Muscle

Jeannine M. Scott,1 Christiana DelloRusso,1 Dennis J. Hartigan-O'Connor,2 Jeffrey S. Chamberlain.1

1Neurology, University of Washington School of Medicine, Seattle, WA, United States;
2Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, United States

Our research has been aimed at exploring the potential of viral vectors as gene therapy tools for Duchenne muscular dystrophy. For this work, we have been developing the high-capacity, or [dsquote]gutted[dsquote] adenoviral vector system which provides a beneficial combination of high-capacity, high transduction efficiency in muscle tissue and vector persistence. In addition to these features, a critical component of any gene therapy protocol will be to monitor the contribution of the host immune system to maintenance or rejection of the therapeutic agent.

We have previously reported functional correction of the dystrophic phenotype in one year-old mdx mice following a single intramuscular injection of gutted adenoviral vectors containing the full-length cDNA for either murine or human dystrophin under the control of a muscle specific promoter. Although expression persisted greater than four weeks, we observed immune cell infiltration in the muscle injected with human dystrophin, most likely due to the fact that this protein is a foreign antigen in the mdx mouse. Because gene therapy patients may also be immunologically naive with respect to dystrophin, we have constructed a gutted adenovirus containing a full-length cDNA encoding utrophin, a functional homologue of dystrophin which is present in mdx mice. We reason that utrophin will not be immunogenic in these animals and the vectors may potentially persist longer than the dystrophin constructs. Indeed, with this vector, we have achieved high-level expression in mdx muscles without any evidence of immune rejection. These vectors containing either human dystrophin or mouse utrophin are being compared with respect to expression levels, persistence, immunogenicity, and effect on muscle function.


Abstract No. 1124

Development of a Small Animal Model of Circulatory Isolation To Promote Efficient Vector Access to Skeletal Muscle in the Pelvic Girdle and Lower Limbs

Leonard T. Su,1 James A. Lesniewski,1 James M. Burkman,1 Zhonglin Wang,1 Xiang G. Zheng,1 Charles R. Bridges,1 Hansell H. Stedman.1

1Department of Surgery, University of Pennsylvania Health System, Philadelphia, PA, United States

A significant obstacle to gene therapy for muscular dystrophy has been the achievement of efficient gene delivery to skeletal muscle on a regional or systemic scale. Intramuscular administration is limited by local diffusion while simple intravascular approaches are extremely inefficient. Previously, we have described highly effective gene transfer in the hindlimbs of rats and cardiomyopathic hamsters, utilizing a method of isolated limb perfusion with surgical placement of hindlimb tourniquets, vasodilatation with papavarine, followed by endothelial permeabilization with histamine. We now introduce a model system for the study of systemic gene delivery to the entire hindbody in rats. The infrarenal aorta and inferior vena cava are isolated and overlapping intramuscular tourniquets are place at this level to achieve vascular isolation in both flanks, the spine and the colon. The effect is complete isolation of the lower torso, pelvis and bilateral hindlimbs from systemic flow. Cannulation of one saphenous artery and a contralateral epigastric vein allows for ateriovenous perfusion of the lower hemibody. Papavarine and histamine are infused along with marker dye or marker virus, which is followed by a volume of saline. The effect of histamine exposure with the infusion of a large "chase" volume is a markedly edematous hindbody, clearly distinguishable from the grossly normal tissue proximal to the tourniquets. No major physiologic changes are seen in blood pressure, heart rate or respirations. Following a dwell period the hindbody is flushed out with saline injected intra-arterially while histamine and papavarine are returned through the venous catheter. After removal of the tourniquets, again no change in physiologic parameters is seen. The animals tolerate the procedure well showing that vascular isolation can be simultaneously achieved in the pelvic girdle and both hindlimbs. The model also shows that the approach to endothelial permeabilization used in our isolated hindlimb model can be extended to a larger volume without untoward systemic effects, as long as the agents are flushed from the isolated circulation. Varying parameters including vascular mediator dosage and degree of hydrostatic pressures are being tested. Application of this model has important implications for the treatment of muscular dystrophies in view of their severe involvement of the proximal musculature. Presentation will encompass our interim progress with the model system. All methods in this model are approved under the University of Pennsylvania Institutional Animal Care and Use Committee.

Abstract No. 1135

Dystrophin Delivery in Mdx Mice Via Retrovirally Transduced Early Myogenic Progenitor Cells

Makoto Ikezawa,1,4 Hairong Peng,2 Zhuqing Qu-Petersen,2 Xiao Xiao,3 Ryan Pruchnic,2 Baohong Cao,1 Shigemi Kimura,1,4 Teruhisa Miike,4 Johnny Huard.1,2

1Growth and Development Laboratory, Children's Hospital of Pittsburgh, Pittsburgh, PA, United States;
2Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, United States;
3Molecular Genetics & Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States;
4Department of Child Development, Kumamoto University, Kumamoto, Kumamoto, Japan

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disease caused by loss of dystrophin in skeletal muscle. Dystrophin gene transfer to skeletal muscle has been hindered by two major limitations: the immune rejection problems related to viral infection and the inability of viral vectors to transduce mature myofibers. We have recently investigated the use of a retroviral vector to deliver dystrophin in dystrophin-deficient mdx mice with ex vivo approach based on muscle derived, early myogenic progenitor cells. Retrovirus carrying human mini-dystrophin (RetroDys3999) was engineered and early myogenic progenitor cells were obtained from a mdx mouse (mdx pp#6) using the preplate technique. Mdx pp#6 cells were stably transduced with RetroDys3999 (mdx pp#6/Dys3999) to express dystrophin. Even after stable transduction, those cells expressed stem cell markers CD34 and Sca1, suggesting that cells preserved their stem cell potential after transduction. Mdx pp#6/Dys3999 cells injected into mdx mouse skeletal muscle were capable of delivering dystrophin. The number of dystrophin positive myofibers was stable for up to 12 weeks after injection. At 2 and 4 weeks after injection, CD4 and CD8a positive lymphocyte infiltration was detected in the injected muscle. These data suggest that even with cells obtained from the same strain of recipient mouse and transduced with retroviral vector, which do not encode any viral proteins, an immune reaction against the injected cells still occurred. The cause of immune rejection was unclear, but the dystrophin protein may be responsible for the immune reaction.

Abstract No. 1130

Muscle Cell Differentiation and Intracellular Localization of Dystrophin-EGFP Fusion Proteins by Confocal Microscopy

Romesh A. Draviam,1 Bing Wang,1 Xiao Xiao.1

1Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

Dystrophin, as a critical muscle cell structure protein, is specifically expressed in differentiated myotubes and myofibers. Deficiency of dystrophin protein in humans by genetic mutation causes Duchenne muscular dystrophy (DMD), a disabling and lethal disease without treatment currently available. To study the intracellular localization of the dystrophin protein during muscle cell differentiation, we have generated two fusion protin constructs, where the Enhanced Green Fluorescent Protein (EGFP) and a mini-dystrophin gene are fused either at the N- or C- terminus of the dystrophin protein, respectively.[br]To test the gene expression, the N- or C- terminus fusion constructs were transfected into 293 human kidney cells and C2C12 mouse myoblast progenitor cells. Both cell lines showed appropriate expression 24 hours post transfection. The clones were then transfected into another myoblast cell line (C57). Appropriate expression and localization at the plasma membrane was observed in fully differentiated myotubes 4 days after transfection. Finally plasmid DNA from both constructs were injected into 6 month mdx (dystrophin deficient) mice. One week after injection appropriate localization of both constructs was confirmed in vivo.[br]Confocal microscopy study showed that the EGFP signals were correctly localized to the plasma membrane in differentiated muscle cells, whereas a random distribution of the EGFP signals were observed in the undifferentiated myoblast cells. Immunofluorescent staining with a human-dystrophin-specific antibody revealed the co-localization of the human mini-dystrophin with the EGFP. As a control, an EGFP-dystrophin construct that had large-deletions in the dystrophin region did not show membrane localization in either undifferentiated of differentiated muscle cells (in vitro and invivo). Thus, we are able to show the biochemical functionality of the fusion proteins, which could be used as a powerful tool to study muscle cell differentiation and mini-dystrophin functions.


Abstract No. 257

Dual Vectors Expressing Murine Factor VIII Result in Sustained Correction of Hemophilia a Mice

Cathryn Mah,1 Rita Sarkar,2 Irene Zolotukhin,1 Mary Schleissing,1 Xiao Xiao,3 Haig H. Kazazian,2 Barry J. Byrne.1

1Powell Gene Therapy Center and Department of Pediatrics, University of Florida, Gainesville, FL, United States;
2Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, United States;
3Department of Molecular Genetics and Biochemistry and Duchenne Muscular Dystrophy Research Center, University of Pittsburgh, Pittsburgh, PA

Hemophilia A is a sex-linked disorder that results from a deficiency of functional factor VIII protein and is currently treated with protein replacement therapies. Gene therapy may provide an alternate treatment modality in which certain limitations of factor VIII protein replacement therapies can be overcome. Within the past decade, adeno-associated virus (AAV)-based gene therapy efforts have come to the forefront of novel therapeutics. In this work, we demonstrate therapeutic levels of recombinant AAV2-mediated expression of murine factor VIII (mFVIII) in a mouse model of hemophilia A. A dual-vector approach was employed in which the heavy and light chains of the mFVIII gene were packaged in two separate rAAV vectors and co-administered either intramuscularly or intravenously to hemophilia A mice. From in vitro work, it was determined that co-infection with both heavy chain and light chain vectors is required for functional factor VIII formation as heterodimerization of the heavy and light chains occurs intracellularly. In vivo, therapeutic levels of factor VIII expression were seen in the mouse model of hemophilia A throughout the duration of the study of 22 weeks, with maximal average expression at 31.4% and 29% of normal, from intravenous and intramuscular administration, respectively, as determined by Coatest assay. Western analysis of plasma cryoprecipitate as well as immunostaining of injection sites using an anti-murine factor VIII light chain antibody also confirmed the expression of factor VIII protein. Since the murine form of the gene was used in the mouse model, less than one Bethesda Unit of inhibitory antibodies was noted. This work demonstrates the feasibility of using rAAV vectors for the long-term treatment of hemophilia A. This work is supported by the National Hemophilia Foundation.


Abstract No. 42

IL-6 Activates the Utrophin Gene Transcription through Promoter A in Neonatal mdx Skeletal Muscles

Yuka Itoh,1 Keita Fujimori,1 Yuko Miyagoe-Suzuki,1 Shin'ichi Takeda.1

1Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disorder caused by a defect in the DMD gene. Utrophin is an autosomal homologue of the DMD gene product, dystrophin, and over-expression of utrophin is expected to compensate the defect of dystrophin and provide a new therapeutic approach for DMD. We previously reported that utrophin was up-regulated on the extrasynaptic sarcolemma in dystrophin-deficient mdx mice through immune responses against a b-galactosidase expressing adenovirus vector, AxCALacZ (Yamamoto et al., Hum Gene Ther 11: 669-680, 2000). We further elucidated the hypothesis that some particular cytokines have been concerned in the over-expression of utrophin under the influence of adenoviral infection. Among the cytokines tested, injection of recombinant IL-6 (rIL-6) successfully increased expression of endogenous utrophin in neonatal mdx skeletal muscle. Importantly, the expression of utrophin was observed in the sarcolemma of mature myofibers as well as in that of regenerating myofibers (Fujimori et al., Hum Gene Ther, in press). We next tried to investigate the molecular mechanism of over-expression of utrophin by administration of rIL-6 and are particularly interested in whether over-expression of utrophin was occurred in transcriptional level or not. Dr. Davies' group had previously identified promoter A and B with their unique 1 st exons of the human/murine utrophin gene. We also noticed another murine utrophin transcript with unique 5'-end and found that new exon 1, designated as exon 1A', was situated between exon 1A and 1B. We then tried to quantify each of three utrophin transcripts by using TaqMan RT-PCR after the injection of IL-6 or AxCALacZ. We daily injected rIL-6 at a dose of 800 ng/day into tibialis anterior (TA) muscles of 2-week-old mdx for 5 days or AxCALacZ into 1-week-old mdx TA muscles, extracted total RNA from pooled 5-6 TA muscles. IL-6 can transiently increase one of three utrophin transcripts, A-utrophin, but AxCALacZ can induce three utrophin mRNA expression until 4weeks after the injection. These results suggested that IL-6 might induce transcription of utrophin through activation of promoter A. On the other hand, stabilization of utrophin mRNA would be probably concerned in the over-expression utrophin by administration of AxCALacZ. Further investigation regarding intracellular signaling pathway of IL-6 and elucidation of stabilizing factor of utrophin mRNA would provide a new aspect of therapy for DMD patients.

Abstract No. 446

Mokola Pseudotyped HIV Vector Expressing Dystrophin Can Protect mdx Mice from Exercise-Induced Necrosis of Muscle Fibers

Jean-Pierre Louboutin,1 Gary Kobinger,1 Tippi McKenzie,2 Alan W. Flake,2 James M. Wilson.1

1Institute for Human Gene Therapy, University of Pennsylvania, and the Wistar Institute, Philadelphia, PA, United States;
2Department of Surgery, Children's Hospital of Philadelphia, Philadelphia, PA, United States

A deficiency of dystrophin in Duchenne Muscular Dystrophy (DMD) results in a progressive degeneration of striated muscle leading to premature death. The mdx mouse has a point mutation in the dystrophin gene and shows similarities with DMD (muscle necrosis/regeneration, elevated CK). VSV-G pseudotyped vector has been proposed for in vivo gene transfer in different applications. However, the low transduction efficiency observed with VSV-G-pseudotyped HIV vector in skeletal muscle has precluded its application in gene therapy of DMD. In an attempt to improve efficiency of transduction with lentiviral vectors, we evaluated constructs pseudotyped with envelopes from MuLV, LCMV, Ebola, Rabies, Mokola, or VSV-G (Env-HIV-LacZ). These experiments revealed that Mokola and Ebola pseudotyped vectors efficiently transduced muscle fibers and satellite cells, respectively. Administration of Ebola- and Mokola-pseudotyped HIV expressing a 6.3 Kb truncated dystrophin gene was performed in the Tibialis Anterior (TA) of adult mdx mice. The contralateral TA muscle was not injected with vector to serve as an internal control. TA muscles were harvested 4 months after the injection of the vector. Prior to harvest, the mice were subject to exercise to induce putative injury and were intravenously injected with Evans Blue, a dye that penetrates degenerating muscle fibers. Dystrophin-positive fibers were observed in the injected TA muscles injected with Mokola-dystrophin (Mok group) but not in muscles injected with Ebola-dystrophin (EboZ group). There were less than 1% revertant fibers in the contralateral uninjected mdx TA muscles. Dystrophin-associated glycoproteins (DAGs) expression was observed in the TA of the Mok group, but not of the EboZ group or the control group. The numbers of calcium positive (alizarin-red S) and Evans blue positive fibers were significantly lower in the Mok group compared to the Ebo Z group and uninjected muscles. Significantly less necrotic fibers and less complement membrane attack complex positive fibers (MAC) were observed in the Mok group as compared to the EboZ group and uninjected contralateral muscles. These results demonstrate that Mokola pseudotyped HIV vector-mediated gene transfer of dystrophin can restore the subsarcolemmal DAGs and dystrophin and can prevent exercise-induced necrosis of muscle fibers in mdx mice.


Abstract No. 1120

Insertion of Adenovirus E4 Region ORF3 into a Helper-Dependent Fully Gutted Adenoviral Vector: Lack of Influence on Initial Level of Gene Expression and Long-Term Expression in Muscle

Jatinderpal R. Deol,1 Renald Gilbert,1 An-Bang Liu,2 Paul C. Holland,1 Josephine Nalbantoglu,1 George Karpati.1

1Neuromuscular Research, Montreal Neurological Institute, Montreal, Quebec, Canada;
2Department of Neurology, Tzu Chi Medical Center, Hualien, Taiwan

Duchenne muscular dystrophy (DMD) is characterized by necrosis and progressive loss of muscle fibers. DMD patients have a mutation in the gene encoding dystrophin, a large membrane-associated cytoskeletal protein on the cytoplasmic side of the sarcolemma. Replacement gene therapy using fully deleted adenoviral vectors shows great potential for the eventual treatment of DMD and other genetic diseases. These vectors are less immunogenic than their predecessors and have the capacity to carry large cDNA inserts such as that of the full-length dystrophin (12 kb). However, the lack of viral genes results in a weak and declining transgene expression in muscle (Hum. Gene Ther. 12: 1741, 2001). Findings in the lung and liver have shown that the adenoviral E4 region, in particular open reading frame 3 (ORF3) to contribute to the maintenance of transgene expression (J. Virol. 73: 8308, 1999) (Hum. Gene Ther. 10: 1833, 1999). We constructed an adenovirus, AdCBDysORF3, in which E4 ORF3 was reintroduced into a fully gutted adenovirus along with full-length dystrophin cDNA controlled by a strong hybrid promoter (CB). Dystrophin levels produced by AdCBDysORF3 were found to be modest at 10 days and not maintained in dystrophin-deficient mdx mice (animal model for DMD). Neonatal muscle sections revealed an average of 120 positive dystrophin fibers at ten days. That number peaked at thirty days with 180 dystrophin-positive fibers before decreasing significantly to 40 fibers at ninety days. Increasing the expression of the primary adenovirus receptor in muscle resulted in a higher initial dystrophin expression in adult mdx mice. When AdCBDysORF3 was injected at the same titer in adult mdx mice transgenic for the coxsackie and adenoviral receptor (CAR), the number of positive fibers tripled in comparison to the non-transgenic littermates. Furthermore, when AdCBDysORF3 was injected into neonatal mdxmuscle mixed with another partially deleted adenovirus containing intact E1B and the full E4 region, the number of dystrophin positive fibers rose ten-fold as compared to AdCBDysORF3 alone. Assuming the entire E4 region can be substituted by ORF3, this result suggests that the combination of the E1B region and ORF3 may be beneficial for initial transgene expression from a gutted adenovirus.


Abstract No. 440

Transduction of Satellite Cells after In Utero Intramuscular Injection of Mokola and Ebola Pseudotyped Lentiviral Vectors

Tippi C. MacKenzie,1 Gary P. Kobinger,2 Jean-Pierre Louboutin,2 Antoneta Radu,1 James M. Wilson,2 Alan W. Flake.1

1Children's Institute for Surgical Science, Children's Hospital of Philadelphia, Philadelphia, PA, United States;
2The Wistar Institute, Philadelphia, PA, United States

Lentiviral vectors constructed using envelope glycoproteins from other viruses may have improved transduction efficiencies for specific targets, such as muscle. We have previously reported long-term expression of lacZ in skeletal and cardiac myocytes after in utero intramuscular injection of Mokola and Ebola pseudotyped vectors, with greater efficiency compared to the VSV-G pseudotype. In these experiments, in addition to transduced myocytes, we also observed small transduced cells at the periphery of the muscle fibers, which had the morphology of satellite cells, or muscle stem cells. We have now confirmed that these are satellite cells using high resolution plastic sectioning, immunofluorescence, and electron microscopy. All vectors were constructed with a transfer vector containing CMV-lacZ. Balb/c fetuses at 14-15 days' gestation were injected with Mokola or Ebola pseudotyped lentiviral vectors in the left hindlimb using pulled glass micropipettes. Animals were allowed to be born and sacrificed at various time points following injection. Muscles were processed for cryosectioning, high resolution plastic sectioning (1-2 um), or transmission electron microscopy (EM). Light microscopy with plastic sections demonstrated transduced cells at the periphery of the muscle fibers, with the typical morphology of satellite cells, after in utero injection of Ebola and Mokola pseudotypes. Double immunofluorescence for b-galactosidase and markers of satellite cells confirmed these findings. In order to localize areas of efficient transduction for further processing for EM, muscles were fixed and X-gal stained en block, then processed for EM. Consecutive thick and thin sections were taken in order to view the same cells under both light microscopy and EM. X-gal deposits were visualized as black crystals under EM in cells which appeared blue under light microscopy, and only in those cells, confirming the specificity of black crystals as a marker of transduced cells at the EM level. Muscle specimens from animals injected with either Mokola or Ebola pseudotyped lentiviral vectors demonstrated transduced satellite cells at the EM level (Figure 1 demonstrates a satellite cell on EM with X-gal crystals, adjacent to a transduced myocyte). This is the first study that clearly demonstrates transduction of muscle satellite cells following viral vector mediated gene transfer. Our results show that Mokola and Ebola pseudotyped lentiviral vectors demonstrate efficiency for satellite cell transduction after in utero intramuscular injection. These findings may have important implications for gene therapy strategies directed toward muscular dystrophy.

Abstract No. 1116

Murine In Utero Gene Delivery by Intramuscular Injection of RGD-Targeted and Untargeted High Capacity Adenovirus

Roberto Bilbao,1 Daniel P. Reay,1 Tiffany H. Fitzsimmons,1 Laura Goldberg,1 Volker Biermann,2 Stefan Kochanek,2 Paula R. Clemens.1,3,4

1Department of Neurology, University of Pittsburgh, Pittsburgh, PA, United States;
2Center for Molecular Medicine, University of Cologne, Cologne, Germany;
3Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States;
4Neurology Service, Department of Veterans Affairs Medical Center, Pittsburgh, PA, United States

In utero gene delivery holds promise for the treatment of hereditary diseases such as Duchenne muscular dystrophy. Among several viral and non-viral vectors for gene therapy, high capacity adenovirus (HC-Ad) is attractive for gene therapy because this vector has a large insert capacity, low immunogenicity and features long transgene expression. Retargeting HC-Ad by inserting the Arg-Gly-Asp (RGD) peptide in the fiber protein of virus was shown to increase adenoviral gene transfer efficiency to smooth muscle cells. The RGD peptide motif binds specifically to integrins that are found in muscle of embryos at 16 days gestation (E-16). We have investigated if targeting of HC-Ad can increase the gene transfer efficiency to muscle in utero. To our knowledge, these data represent the first study where targeted HC-Ad has been used to deliver genes in utero.

To achieve this, primary myoblasts isolated from E-16 embryos and cell lines with high (A549 lung carcinoma cells) and low (NIH3T3 fibroblasts) levels of CAR receptor were infected with RGD-modified HC-Ad, unmodified HC-Ad and first generation adenovirus at different MOIs. All three vectors encoded the lacZ transgene driven by the cytomegalovirus (CMV) promoter. For in vivo studies, we injected the three vectors intramuscularly to E-16 fetuses to test their gene transfer efficiency to muscle. Each vector was diluted to 1.8x107pfu/embryo and injected in the embryo leg. Cells or tissues (muscle, heart, liver, gut, lung) were collected two days after infection and analyzed for β-galactosidase expression by the o-nitrophenyl-β-D-galactopyranoside (ONPG) assay.

In vitro, unmodified HC-Ad was the most efficient vector for the transduction of primary embryonic myoblasts and A549 cells. Higher transduction with RGD modified HC-Ad was only observed in NIH/3T3 fibroblasts. Intramuscular in utero delivery of the three different vectors resulted in a significantly higher transgene expression in those pups that were injected with the unmodified HC-AD as compared to those that received the RGD-modified HC-Ad or the first generation adenovirus. In contrast to the leg muscle expression, in heart and liver, we did not find differences in gene expression between vectors.

Our results suggest that 1) HC-Ad is a more efficient vector than first generation adenovirus for in utero gene delivery, and 2) RGD-modified HC-Ad was not superior to unmodified HC-Ad infection of muscle cells both in vitro and in vivo. Our ongoing research is focused on determining the mechanisms by which RGD targeting alters HC-Ad transduction in muscle in utero and identifying other candidates for HC-Ad targeting to embryonic muscle.