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Successful AAV vector_mediated gene transfer into canine skeletal muscle required suppression of excess immune responses
MUSCLE 244. Successful AAV Vector-Mediated Gene Transfer into Canine Skeletal Muscle Required Suppression of Excess Immune Responses
245. Muscle-Derived Stem Cells Display an Extended, but Not Unlimited, Expansion Capability: Implication for Muscle Regeneration
Katsutoshi Yuasa,1 Madoka Yoshimura,1 Nobuyuki Urasawa,1 Katsujiro Sato,1 Yuko Miyagoe-Suzuki,1 John McC Howell,2 Shin’ichi Takeda.1 1 Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; 2Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, WA, Australia.
Bridget M. Deasy,1,2 Burhan M. Gharaibeh,2 Michele Jones,2 Michael A. Lucas,1,2 Johnny Huard.1,2,3 1 Bioengineering, University of Pittsburgh; 2Growth and Development Laboratory, Children’s Hospital of Pittsburgh; 3 Molecular Genetics & Biochemistry and Orthopedic Surgery, University of Pittsburgh Medical Center.
Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by a mutation 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. To find a short but functional dystrophin cDNA, we have previously constructed three micro-dystrophin cDNAs, and generated transgenic (Tg) dystrophin-deficient mdx mice expressing micro-dystrophin. Among them, CS1-Tg mdx mice showed lowest levels of serum creatine kinase, complete amelioration of muscle pathology, and nearly full restoration of contractile force (BBRC. 293:1265, 2002). We also showed that muscle-specific MCK promoter in AAV vector could drive longer expression of the LacZ gene than the CMV promoter in skeletal muscle (Gene Ther. 23:1576, 2002). Furthermore, we constructed the AAV2 vector expressing ∆CS1 micro-dystrophin driven by MCK promoter, and demonstrated that AAV vectormediated ∆CS1 transfer could ameliorate dystrophic phenotypes in mdx muscles (7th ASGT Annual Meeting 2004, in submission). For the application of this strategy to DMD patients, however, it is necessary to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We recently established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp. Anim. 52: 93, 2003). When the AAV vector encoding the LacZ gene driven by a CMV promoter was introduced into skeletal muscles of dogs, β-galactosidase (β-gal) was expressed only in few fibers of injected muscle after 2 weeks of injection. No β-gal-positive fiver was detected in canine muscle at 4 and 8 weeks post-injection. Instead, large numbers of mononuclear cells appeared around β-gal-expressing fibers in injected muscle. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of AAV vector, we examined viral infectivity, cytotoxicity and immune responses. First, we infected AAV vector into canine primary myotubes. This in vitro study showed that AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particle elicit cytotoxicity or not. When the AAV vector expressing no transgene 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-β-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 considerably improved transduction efficiency by an AAV vector introduction in canine muscle. These results suggested that AAV vector-mediated gene transfer elicited stronger immune responses in canine muscle, and it was necessary to know the molecular background of excess immune responses and to find the way to minimize and suppress immune responses.
S94
Stem cells are frequently considered the optimal cell type for regenerative cell–based therapies; however they generally represent a small fraction of cells isolated from a biopsy or other cell source. Ex vivo cell expansion is a necessary step to obtain clinically relevant numbers of cells. In addition, stem cells are often theorized as cells with unlimited long-term expansion potential. The purpose of this study is to test the long-term expansion capability of a population of muscle-derived stem cells. We first examined the proliferation kinetics of murine muscle-derived stem cells (MDSCs) to determine if they obey Hayflick’s limit. We determined that these cells can be expanded for more than 300 population doublings (PDs) with no indications of replicative senescence. Next we examined how the molecular and behavioral stem cell phenotype, including the regenerative capacity, changes over time. We find that the MDSC population continues to maintain a relatively low level of desmin expression (<30%), and a high level of stem cell antigen 1 (Sca-1) expression (>65%) throughout the expansion. We observe that up to 200 PDs the MDSCs readily differentiate to form multinucleated myotubes, however expansion beyond 200 PDs leads to a decline in the number of cells entering the post-mitotic differentiated state. Remarkably, MDSC are capable of regenerating dystrophin expressing muscle fibers upon implantation in mdx muscular dystrophy model even after 200 population doublings. However, expansion beyond 200 PDs resulted in a subsequent decline in regeneration efficiency. Observed phenotypic changes highlight the inevitable aging of cells that results from cell expansion. Several findings including loss of contact inhibition, ability to grow on soft agar and an increase in numerical chromosomal abnormalities suggests that the MDSC may have become transformed. While the MDSC demonstrate a highly extended functional lifetime for muscle regeneration, we find that this potential is not unlimited.
246. Gender Differences in Transplantation Efficiency Using Muscle-Derived Stem Cells for Muscular Dystrophy Bridget M. Deasy,1 Aiping Lu,2 Burhan M. Gharaibeh,2 Michele Jones,2 Jessica Tebbets,2 Johhny Huard.1,2,3 1 Bioengineering, University of Pittsburgh, Pittsburgh, PA; 2 Growth and Development Laboratory, Children’s Hospital of Pittsburgh, Pittsburgh, PA; 3Molecular Genetics & Biochemistry and Orthopedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA. Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disease characterized by progressive muscle weakness due to the lack of dystrophin expression at the sarcolemma of muscle fibers. Transplantation of normal myoblasts into diseased muscle provides donor myoblasts that fuse with dystrophic muscle fibers and restore dystrophin. This process enables transient dystrophin delivery and improved strength in the injected dystrophic muscle. However, the approach has limitations, including immune rejection, poor cellular survival rates, and limited dissemination of the donor cells. The outcome of this cell transplantation therapy has been improved in the murine DMD model (mdx) by using muscle-derived stem cells (MDSCs). This enhanced success appears to be attributable to several unique features of stem cells: 1) self-renewal with Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
245. Muscle-Derived Stem Cells Display an Extended, but Not Unlimited, Expansion Capability: Implication for Muscle Regeneration
Katsutoshi Yuasa,1 Madoka Yoshimura,1 Nobuyuki Urasawa,1 Katsujiro Sato,1 Yuko Miyagoe-Suzuki,1 John McC Howell,2 Shin’ichi Takeda.1 1 Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; 2Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, WA, Australia.
Bridget M. Deasy,1,2 Burhan M. Gharaibeh,2 Michele Jones,2 Michael A. Lucas,1,2 Johnny Huard.1,2,3 1 Bioengineering, University of Pittsburgh; 2Growth and Development Laboratory, Children’s Hospital of Pittsburgh; 3 Molecular Genetics & Biochemistry and Orthopedic Surgery, University of Pittsburgh Medical Center.
Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by a mutation 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. To find a short but functional dystrophin cDNA, we have previously constructed three micro-dystrophin cDNAs, and generated transgenic (Tg) dystrophin-deficient mdx mice expressing micro-dystrophin. Among them, CS1-Tg mdx mice showed lowest levels of serum creatine kinase, complete amelioration of muscle pathology, and nearly full restoration of contractile force (BBRC. 293:1265, 2002). We also showed that muscle-specific MCK promoter in AAV vector could drive longer expression of the LacZ gene than the CMV promoter in skeletal muscle (Gene Ther. 23:1576, 2002). Furthermore, we constructed the AAV2 vector expressing ∆CS1 micro-dystrophin driven by MCK promoter, and demonstrated that AAV vectormediated ∆CS1 transfer could ameliorate dystrophic phenotypes in mdx muscles (7th ASGT Annual Meeting 2004, in submission). For the application of this strategy to DMD patients, however, it is necessary to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We recently established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp. Anim. 52: 93, 2003). When the AAV vector encoding the LacZ gene driven by a CMV promoter was introduced into skeletal muscles of dogs, β-galactosidase (β-gal) was expressed only in few fibers of injected muscle after 2 weeks of injection. No β-gal-positive fiver was detected in canine muscle at 4 and 8 weeks post-injection. Instead, large numbers of mononuclear cells appeared around β-gal-expressing fibers in injected muscle. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of AAV vector, we examined viral infectivity, cytotoxicity and immune responses. First, we infected AAV vector into canine primary myotubes. This in vitro study showed that AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particle elicit cytotoxicity or not. When the AAV vector expressing no transgene 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-β-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 considerably improved transduction efficiency by an AAV vector introduction in canine muscle. These results suggested that AAV vector-mediated gene transfer elicited stronger immune responses in canine muscle, and it was necessary to know the molecular background of excess immune responses and to find the way to minimize and suppress immune responses.
S94
Stem cells are frequently considered the optimal cell type for regenerative cell–based therapies; however they generally represent a small fraction of cells isolated from a biopsy or other cell source. Ex vivo cell expansion is a necessary step to obtain clinically relevant numbers of cells. In addition, stem cells are often theorized as cells with unlimited long-term expansion potential. The purpose of this study is to test the long-term expansion capability of a population of muscle-derived stem cells. We first examined the proliferation kinetics of murine muscle-derived stem cells (MDSCs) to determine if they obey Hayflick’s limit. We determined that these cells can be expanded for more than 300 population doublings (PDs) with no indications of replicative senescence. Next we examined how the molecular and behavioral stem cell phenotype, including the regenerative capacity, changes over time. We find that the MDSC population continues to maintain a relatively low level of desmin expression (<30%), and a high level of stem cell antigen 1 (Sca-1) expression (>65%) throughout the expansion. We observe that up to 200 PDs the MDSCs readily differentiate to form multinucleated myotubes, however expansion beyond 200 PDs leads to a decline in the number of cells entering the post-mitotic differentiated state. Remarkably, MDSC are capable of regenerating dystrophin expressing muscle fibers upon implantation in mdx muscular dystrophy model even after 200 population doublings. However, expansion beyond 200 PDs resulted in a subsequent decline in regeneration efficiency. Observed phenotypic changes highlight the inevitable aging of cells that results from cell expansion. Several findings including loss of contact inhibition, ability to grow on soft agar and an increase in numerical chromosomal abnormalities suggests that the MDSC may have become transformed. While the MDSC demonstrate a highly extended functional lifetime for muscle regeneration, we find that this potential is not unlimited.
246. Gender Differences in Transplantation Efficiency Using Muscle-Derived Stem Cells for Muscular Dystrophy Bridget M. Deasy,1 Aiping Lu,2 Burhan M. Gharaibeh,2 Michele Jones,2 Jessica Tebbets,2 Johhny Huard.1,2,3 1 Bioengineering, University of Pittsburgh, Pittsburgh, PA; 2 Growth and Development Laboratory, Children’s Hospital of Pittsburgh, Pittsburgh, PA; 3Molecular Genetics & Biochemistry and Orthopedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA. Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disease characterized by progressive muscle weakness due to the lack of dystrophin expression at the sarcolemma of muscle fibers. Transplantation of normal myoblasts into diseased muscle provides donor myoblasts that fuse with dystrophic muscle fibers and restore dystrophin. This process enables transient dystrophin delivery and improved strength in the injected dystrophic muscle. However, the approach has limitations, including immune rejection, poor cellular survival rates, and limited dissemination of the donor cells. The outcome of this cell transplantation therapy has been improved in the murine DMD model (mdx) by using muscle-derived stem cells (MDSCs). This enhanced success appears to be attributable to several unique features of stem cells: 1) self-renewal with Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy