- Email: [email protected]
455. Characterization of A20 as an Inhibitor of NF-κB Activation in Dystrophic Mice
MUSCULO-SKELETAL GENE & CELL THERAPY II polyclonally stimulated T-cells (p<0,05) in a 1:2 ratio. Insertion of MOG-reactive CAR into MSC made CNS-specic MSCs that retained their capacity to signicantly suppress T-cell proliferation (p<0,001) in a 1:2 ratio. Further, when adding MOG+ cells to the co cultures the engineered Tregs and MSCs (p<0,001) were still suppressive. Furthermore, engineered Tregs and MSCs were able to locate to and transmigrate into the brain of mice 10 days post systemical i.p. injection. In conclusion, genetically engineered suppressor cells are able to localize to the CNS and to suppress the proliferation of activated T-cells.
Musculo-skeletal Gene & Cell Therapy II 454. First Generation AAVmicroutrophin Vector Infused into the Isolated Pelvic Limb of a Canine Model for Duchenne Muscular Dystrophy
Mihail Petrov,1 Marilyn Mitchell,1 Alock Malik,1 Andy Mead,1 Frederick Balzer,1 Leonard Su,1 Jacqueline Farag,1 Benjamin Kozyak,1 Kapil Gopal,1 Charles Bridges,1 Janet Bogan,2 Martin Childers,3 Joe Kornegay,2 Hansell Stedman.1 1 Surgery, University of Pennsylvania, Philadelphia, PA; 2University of North Carolina, Chapel Hill, NC; 3Wake Forest Univ., Winston Salem, NC. Germline and somatic gene transfer of internally deleted dystrophin and utrophin coding sequences into dystrophic mice has provided evidence for phenotypic amelioration. Myotrophic AAV vectors have been shown to be amenable to a vascular route of administration, suggesting that partial phenotypic correction should be feasible in a large animal disease model. To this end, we initially injected the isolated pelvic limbs of dogs hemi- or homozygous for the GRMD mutation (dystrophin intron 6 splice acceptor site) with a “rst generation” AAV6 vector containing a constitutive promoter/enhancer driving transcription of a “microutrophin” cassette based on the wild type cDNA sequence. The afferent, transvenular extravasation route of administration used recapitulates that previously shown to transduce essentially 100% of the skeletal muscle bers in the mature canine leg (Su, Gopal, et al, 2005). A proportion of the dogs underwent transient single agent immunosuppression using a protocol previously shown to prevent inhibitory antibody formation in a canine model for hemophilia B (Arruda, Stedman, et al, 2005). Dogs were injected with vector at one of three doses. Follow up studies for force transduction were performed by a group of investigators blinded as to the AAV dose, identity of the injected limb, and the presence or absence of prior immunosuppression. In a group of dogs receiving the highest dose of AAV.utrophin-1 (10E13.5vg/kg), the ratio of torque developed by the treated vs. untreated limb is 1.07+/-.2. Five of six dogs in this group showed exion strength improvement on the treated side. If one disallows the data from the one confounding dog on the grounds that a minor technical problem occurred (electrode migration during force transduction, as suspected from the discrepancy between serial measurements in this dog) the average ratio among remaining dogs is 1.15+/-.08. Importantly, with this combination of treatment and immunosuppressive regimen the muscles did not become weaker, as one might have expected with an AAV-induced myositis. Interestingly, data on other non-immunosuppressed groups of dogs suggest that the vector might have caused a subclinical myositis. Among dystrophic dogs receiving even lower doses of AAV.utrophin-1 without cyclophosphamide, the ratio of treated to untreated limb torque was 0.94+/-0.05. Moreover, among non-dystrophic dogs receiving the unrelated AAV.F.IX (containing a “self” transgene) without immunosuppression, the ratio of treated to untreated limb strength was 0.85+/-0.06. In conjunction with emerging trends in AAV-based clinical investigation, these observations heighten the impetus to formally address the prevention of cellular immune response directed against input AAV capsid antigens. S176
455. Characterization of A20 as an Inhibitor of NF-țB Activation in Dystrophic Mice Rakshita Charan,1 Paula R. Clemens.1,2 Department of Neurology, University of Pittsburgh, Pittsburgh, PA; 2Department of Veterans Affairs Medical Center, Veterans Affairs, Pittsburgh, PA.
1
Of all muscular dystrophies, Duchenne muscular dystrophy (DMD) is the most common affecting about 1 in 3500 male births worldwide. The disease is caused by mutations in the gene dystrophin, the protein product of which is required for muscle structure and stability. Studies suggest that the lack of structural support in dystrophin-decient muscle bers may be responsible for muscle pathology in progressive muscular dystrophy. It is also known that nuclear factor-kappa B (NF-κB), which is a nuclear transcription factor, is up regulated in dystrophic muscle in DMD patients as well as in the mouse model for DMD (mdx). NF-κB regulates several genes responsible for stress responses, cell survival and various inammatory conditions. Thus, the up regulation of this transcription factor is thought to activate protein degradation and cause chronic inammation in skeletal muscle. Furthermore, NF-κB downregulates myogenic regulatory factors and this process likely interferes with muscle regeneration. Attenuating NF-κB activation in these dystrophic mice has been shown to improve muscle stability and strength. Strategies of inhibition of NF-κB activation are being actively pursued as a therapeutic option for DMD. One of the NF-κB pathway attenuators, A20 is a deubiquitinating enzyme, known to inhibit NF-κB activation by deubiquitinating RIP1; ubiquitination of RIP1 is essential for NF-κB activation. Our aim is to characterize A20 in skeletal muscle and establish its role as a potential therapeutic target as attenuator of the NF-κB pathway activation in DMD. We show that blocking of A20 using A20siRNA increases NF-κB activation in mdx as well as control C57BL/10 mice myotubes. We further characterized localization of A20 in muscle and established that A20 is expressed predominantly in fast-twitch muscle bers. Interestingly, we also observed that in mdx muscle, A20 is over-expressed in regenerating bers. To study the localization of A20 through the life of the mdx mouse, we did a time-prole assessment of A20 expression and compared it with control mice. We see an increase in A20 protein expression during the 7-10 week time period in mice, which is correlative of the ages when severe degeneration and regeneration cycles take place in mdx mice. This is the rst observation of a correlation between expression of an NF-κB inhibitor and pathology of DMD. Our studies support the utility of therapeutic manipulation of A20 to promote NF-κB inactivation in dystrophic muscle bers and provide a potential therapy for DMD.
456. Plasticity of Skeletal Muscle Cells during Muscle Injury and Repair – A Dedifferentiation Study
Xiaodong Mu,1,2 Hairong Peng,2 Johnny Huard,2,3 Yong Li.1,2,3,4 The Laboratory of Molecular Pathology, Stem Cell Research Center (S.C.R.C.),, Children’s Hospital of UPMC, Pittsburgh, PA; 2 Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 3Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA; 4Department of Pathology, University of Pittsburgh, Pittsburgh, PA.
1
INTRODUCTION: In the skeletal muscle system of certain amphibians, dedifferentiation occurs after muscle injury and mononuclear cells are released from myobers. These mononuclear cells can then serve as an source of precursor cells for effective muscle regeneration. However, whether a similar dedifferentiation process occurs in mammals remains largely a mystery, and it was generally believed that the main sources of muscle precursors for efcient Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Musculo-skeletal Gene & Cell Therapy II 454. First Generation AAVmicroutrophin Vector Infused into the Isolated Pelvic Limb of a Canine Model for Duchenne Muscular Dystrophy
Mihail Petrov,1 Marilyn Mitchell,1 Alock Malik,1 Andy Mead,1 Frederick Balzer,1 Leonard Su,1 Jacqueline Farag,1 Benjamin Kozyak,1 Kapil Gopal,1 Charles Bridges,1 Janet Bogan,2 Martin Childers,3 Joe Kornegay,2 Hansell Stedman.1 1 Surgery, University of Pennsylvania, Philadelphia, PA; 2University of North Carolina, Chapel Hill, NC; 3Wake Forest Univ., Winston Salem, NC. Germline and somatic gene transfer of internally deleted dystrophin and utrophin coding sequences into dystrophic mice has provided evidence for phenotypic amelioration. Myotrophic AAV vectors have been shown to be amenable to a vascular route of administration, suggesting that partial phenotypic correction should be feasible in a large animal disease model. To this end, we initially injected the isolated pelvic limbs of dogs hemi- or homozygous for the GRMD mutation (dystrophin intron 6 splice acceptor site) with a “rst generation” AAV6 vector containing a constitutive promoter/enhancer driving transcription of a “microutrophin” cassette based on the wild type cDNA sequence. The afferent, transvenular extravasation route of administration used recapitulates that previously shown to transduce essentially 100% of the skeletal muscle bers in the mature canine leg (Su, Gopal, et al, 2005). A proportion of the dogs underwent transient single agent immunosuppression using a protocol previously shown to prevent inhibitory antibody formation in a canine model for hemophilia B (Arruda, Stedman, et al, 2005). Dogs were injected with vector at one of three doses. Follow up studies for force transduction were performed by a group of investigators blinded as to the AAV dose, identity of the injected limb, and the presence or absence of prior immunosuppression. In a group of dogs receiving the highest dose of AAV.utrophin-1 (10E13.5vg/kg), the ratio of torque developed by the treated vs. untreated limb is 1.07+/-.2. Five of six dogs in this group showed exion strength improvement on the treated side. If one disallows the data from the one confounding dog on the grounds that a minor technical problem occurred (electrode migration during force transduction, as suspected from the discrepancy between serial measurements in this dog) the average ratio among remaining dogs is 1.15+/-.08. Importantly, with this combination of treatment and immunosuppressive regimen the muscles did not become weaker, as one might have expected with an AAV-induced myositis. Interestingly, data on other non-immunosuppressed groups of dogs suggest that the vector might have caused a subclinical myositis. Among dystrophic dogs receiving even lower doses of AAV.utrophin-1 without cyclophosphamide, the ratio of treated to untreated limb torque was 0.94+/-0.05. Moreover, among non-dystrophic dogs receiving the unrelated AAV.F.IX (containing a “self” transgene) without immunosuppression, the ratio of treated to untreated limb strength was 0.85+/-0.06. In conjunction with emerging trends in AAV-based clinical investigation, these observations heighten the impetus to formally address the prevention of cellular immune response directed against input AAV capsid antigens. S176
455. Characterization of A20 as an Inhibitor of NF-țB Activation in Dystrophic Mice Rakshita Charan,1 Paula R. Clemens.1,2 Department of Neurology, University of Pittsburgh, Pittsburgh, PA; 2Department of Veterans Affairs Medical Center, Veterans Affairs, Pittsburgh, PA.
1
Of all muscular dystrophies, Duchenne muscular dystrophy (DMD) is the most common affecting about 1 in 3500 male births worldwide. The disease is caused by mutations in the gene dystrophin, the protein product of which is required for muscle structure and stability. Studies suggest that the lack of structural support in dystrophin-decient muscle bers may be responsible for muscle pathology in progressive muscular dystrophy. It is also known that nuclear factor-kappa B (NF-κB), which is a nuclear transcription factor, is up regulated in dystrophic muscle in DMD patients as well as in the mouse model for DMD (mdx). NF-κB regulates several genes responsible for stress responses, cell survival and various inammatory conditions. Thus, the up regulation of this transcription factor is thought to activate protein degradation and cause chronic inammation in skeletal muscle. Furthermore, NF-κB downregulates myogenic regulatory factors and this process likely interferes with muscle regeneration. Attenuating NF-κB activation in these dystrophic mice has been shown to improve muscle stability and strength. Strategies of inhibition of NF-κB activation are being actively pursued as a therapeutic option for DMD. One of the NF-κB pathway attenuators, A20 is a deubiquitinating enzyme, known to inhibit NF-κB activation by deubiquitinating RIP1; ubiquitination of RIP1 is essential for NF-κB activation. Our aim is to characterize A20 in skeletal muscle and establish its role as a potential therapeutic target as attenuator of the NF-κB pathway activation in DMD. We show that blocking of A20 using A20siRNA increases NF-κB activation in mdx as well as control C57BL/10 mice myotubes. We further characterized localization of A20 in muscle and established that A20 is expressed predominantly in fast-twitch muscle bers. Interestingly, we also observed that in mdx muscle, A20 is over-expressed in regenerating bers. To study the localization of A20 through the life of the mdx mouse, we did a time-prole assessment of A20 expression and compared it with control mice. We see an increase in A20 protein expression during the 7-10 week time period in mice, which is correlative of the ages when severe degeneration and regeneration cycles take place in mdx mice. This is the rst observation of a correlation between expression of an NF-κB inhibitor and pathology of DMD. Our studies support the utility of therapeutic manipulation of A20 to promote NF-κB inactivation in dystrophic muscle bers and provide a potential therapy for DMD.
456. Plasticity of Skeletal Muscle Cells during Muscle Injury and Repair – A Dedifferentiation Study
Xiaodong Mu,1,2 Hairong Peng,2 Johnny Huard,2,3 Yong Li.1,2,3,4 The Laboratory of Molecular Pathology, Stem Cell Research Center (S.C.R.C.),, Children’s Hospital of UPMC, Pittsburgh, PA; 2 Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 3Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA; 4Department of Pathology, University of Pittsburgh, Pittsburgh, PA.
1
INTRODUCTION: In the skeletal muscle system of certain amphibians, dedifferentiation occurs after muscle injury and mononuclear cells are released from myobers. These mononuclear cells can then serve as an source of precursor cells for effective muscle regeneration. However, whether a similar dedifferentiation process occurs in mammals remains largely a mystery, and it was generally believed that the main sources of muscle precursors for efcient Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy