- Email: [email protected]
DC190-GAA to Pompe Mice Normalizes Muscle Pathology and Corrects Motor Function Deficits
MUSCLE AND CONNECTIVE TISSUE: MUSCLE sites 4 weeks later. In the biopsies of the cell-grafted sites, there were myofibers expressing donor’s dystrophin in 8 patients. The percentage of myofibers expressing donor’s dystrophin varied from 3.5% to 26%. Evidence of small-myofiber neoformation was observed in some patients. Donor-derived dystrophin transcripts were detected by RT-PCR in the cell-grafted sites in all patients. The protocol of immunosuppression was sufficient to obtain these results, although it is not certain whether acute rejection was efficiently controlled in all the cases. In conclusion, intramuscular allotransplantation of normal MPCs can induce the expression of donor-derived dystrophin in skeletal muscles of DMD patients, although this expression is restricted to the sites of MPC injection. I have a major stock holder in Cellgene Inc.
32. Improvement of Muscle Mass Using shRNA Targeting Myostatin or Activin Receptor IIb Julie Dumonceaux,1 Solenne Marie,1 Luis Garcia.1 Maladie de Duchenne, Genethon, Evry, France.
1
Duchenne Muscular Dystrophy (DMD) is the most severe degenerative disorder of skeletal and cardiac muscle. DMD patients show a progressive muscle weakness which begins in early childhood. Our goal is to stop this continuous muscle wasting. Myostatin is a negative regulator of skeletal muscle mass. This protein binds to its cell-surface receptor (Activin receptor IIb, AcvRIIb) to inhibit both proliferation and differentiation of myoblasts. In vivo, it was shown that myostatin deficient mice show a double mass phenotype compared to wild type animals. Our hypothesis is that inactivation of myostatin function may consequently results in an increase of muscle mass in DMD patients as well as in its murine model, the mdx mouse. We have investigated RNA interference technique to inhibit functions of myostatin. We decided to target AcvRIIb as well as the myostatin. Mice were injected intra muscularly using an AAV carrying the AcvRIIb shRNA or myostatin shRNA. One of the myostatin shRNA inhibits 95% of the mRNA expression. Preliminary results also show that a 50 % down regulation of AcvRIIb in mdx mice results in an 15% increase in muscle mass. The next step is now to combine on a same AAV vector the exon skipping strategy (ie AAV-U7-SD23/BP22) and the inactivation of myostatin function (ie AAV-AcvRIIb shRNA). This vector may serve to both rescue dystrophin and improve muscle mass.
33. Systemic Delivery of AAV8/DC190-GAA to Pompe Mice Normalizes Muscle Pathology and Corrects Motor Function Deficits R. J. Ziegler,1 M. A. Zhao,1 S. D. Bercury,1 J. A. Fidler,1 J. W. Foley,1 D. Armentano,1 S. Ryan,1 T. Taksir,1 D. Griffiths,1 L. S. Shihabuddin,1 R. K. Scheule,1 S. H. Cheng.1 1 Applied Discovery Research, Genzyme Corporation, Framingham, MA. Pompe disease, a lysosomal storage disorder, is generally known for it’s characteristic degenerative myopathy. Massive accumulation of glycogen in lysosomes of striated and smooth muscle due to a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) results in muscle weakness and cardiorespiratory failure. In severely affected patients glycogen also accumulates in the central and peripheral nervous systems. Previously we demonstrated that systemic administration of AAV8/DC190-GAA to Pompe mice generated high circulating levels of GAA and reduced glycogen storage in cardiac and skeletal muscle to near normal levels. The current study was designed to determine if these effects on the viscera would be sufficient to prevent the loss of muscle strength and coordination shown to develop over time in the Pompe mouse. Since the GAA secreted into the blood should not cross the bloodS14
brain barrier, this study should also clarify the functional significance of peripheral and CNS glycogen storage in the Pompe mouse model. Two different concentrations of AAV8/DC190-GAA were injected into the tail vein of 10-week-old male Pompe mice. Treated Pompe mice, as well as vehicle-treated Pompe and B6129SF2/J normal control mice, were tested for their ability to perform in a battery of behavioral tests. Tests designed to measure muscle strength and coordination included accelerating and rocking Rota-rod, wire hang, and foot fault. As expected, the performance of the vehicle-treated Pompe mice declined over time when compared to wild type mice. In contrast the performance of AAV-treated Pompe mice was significantly improved over that of vehicle-treated Pompe mice, and was similar to normal mice. There was no significant difference between the two AAV8 dosing groups in any test except the wire hang. In this test the performance of the lower dose treatment group declined at the later time points. Severely affected patients rapidly accumulate glycogen, and infants are often not diagnosed until they show obvious myotonia. To model the treatment of this patient population, we administered AAV8/DC190-GAA to 10-month-old Pompe mice, an age at which they display significant muscle pathology and motor function decline. Mice treated at this advanced stage of the disease showed a partial recovery of muscle function, but performance was not normalized despite high circulating levels of GAA. We are currently measuring glycogen levels and studying the histopathology of muscle and CNS tissues from the mice in these studies. In summary, we have shown that systemic gene therapy treatment is capable of preventing degenerative myopathy and preserving motor function in Pompe mice, but is less effective in animals with pre-existing pathology. I am an employee of Genzyme Corporation and hold stock in the company.
34. Phi C31 Integrase System Enhances Dystrophin Gene Expression in Skeletal Muscle of Mouse Models for Duchenne Muscular Dystrophy Carmen Bertoni,1 Sohail Jarrahian,2 Thurman M. Wheeler,1 Yining Li,1 Eric C. Olivares,2 Michele P. Calos,2 Thomas A. Rando.1 1 Neurology, Stanford University, Stanford, CA; 2Genetics, Stanford University, Stanford, CA. Duchenne muscular dystrophy (DMD) is caused by lack of dystrophin expression in skeletal muscles. 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. Plasmidbased gene therapies have been shown to be a valid approach to the treatment of a variety of disorders including DMD. Among the limitations of this technology are the restricted distribution of plasmid following direct intramuscular injection, the challenge of achieving widespread plasmid delivery by systemic injection, and the loss of extrachromosomal plasmid over time. In order to address the latter issue, which could prove to be a major limitation of therapeutic efficacy in DMD, we have studied the effects of targeted plasmid integration using a phage integrase (Phi C31) that has been shown to mediate the integration of suitably modified plasmids into the mammalian genome. Using a luciferase expression plasmid, we were able to monitor plasmid gene expression non-invasively in living mice by bioluminescence imaging (BLI). 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 fC31 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 BLIS. Shortly after delivery, the Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
32. Improvement of Muscle Mass Using shRNA Targeting Myostatin or Activin Receptor IIb Julie Dumonceaux,1 Solenne Marie,1 Luis Garcia.1 Maladie de Duchenne, Genethon, Evry, France.
1
Duchenne Muscular Dystrophy (DMD) is the most severe degenerative disorder of skeletal and cardiac muscle. DMD patients show a progressive muscle weakness which begins in early childhood. Our goal is to stop this continuous muscle wasting. Myostatin is a negative regulator of skeletal muscle mass. This protein binds to its cell-surface receptor (Activin receptor IIb, AcvRIIb) to inhibit both proliferation and differentiation of myoblasts. In vivo, it was shown that myostatin deficient mice show a double mass phenotype compared to wild type animals. Our hypothesis is that inactivation of myostatin function may consequently results in an increase of muscle mass in DMD patients as well as in its murine model, the mdx mouse. We have investigated RNA interference technique to inhibit functions of myostatin. We decided to target AcvRIIb as well as the myostatin. Mice were injected intra muscularly using an AAV carrying the AcvRIIb shRNA or myostatin shRNA. One of the myostatin shRNA inhibits 95% of the mRNA expression. Preliminary results also show that a 50 % down regulation of AcvRIIb in mdx mice results in an 15% increase in muscle mass. The next step is now to combine on a same AAV vector the exon skipping strategy (ie AAV-U7-SD23/BP22) and the inactivation of myostatin function (ie AAV-AcvRIIb shRNA). This vector may serve to both rescue dystrophin and improve muscle mass.
33. Systemic Delivery of AAV8/DC190-GAA to Pompe Mice Normalizes Muscle Pathology and Corrects Motor Function Deficits R. J. Ziegler,1 M. A. Zhao,1 S. D. Bercury,1 J. A. Fidler,1 J. W. Foley,1 D. Armentano,1 S. Ryan,1 T. Taksir,1 D. Griffiths,1 L. S. Shihabuddin,1 R. K. Scheule,1 S. H. Cheng.1 1 Applied Discovery Research, Genzyme Corporation, Framingham, MA. Pompe disease, a lysosomal storage disorder, is generally known for it’s characteristic degenerative myopathy. Massive accumulation of glycogen in lysosomes of striated and smooth muscle due to a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) results in muscle weakness and cardiorespiratory failure. In severely affected patients glycogen also accumulates in the central and peripheral nervous systems. Previously we demonstrated that systemic administration of AAV8/DC190-GAA to Pompe mice generated high circulating levels of GAA and reduced glycogen storage in cardiac and skeletal muscle to near normal levels. The current study was designed to determine if these effects on the viscera would be sufficient to prevent the loss of muscle strength and coordination shown to develop over time in the Pompe mouse. Since the GAA secreted into the blood should not cross the bloodS14
brain barrier, this study should also clarify the functional significance of peripheral and CNS glycogen storage in the Pompe mouse model. Two different concentrations of AAV8/DC190-GAA were injected into the tail vein of 10-week-old male Pompe mice. Treated Pompe mice, as well as vehicle-treated Pompe and B6129SF2/J normal control mice, were tested for their ability to perform in a battery of behavioral tests. Tests designed to measure muscle strength and coordination included accelerating and rocking Rota-rod, wire hang, and foot fault. As expected, the performance of the vehicle-treated Pompe mice declined over time when compared to wild type mice. In contrast the performance of AAV-treated Pompe mice was significantly improved over that of vehicle-treated Pompe mice, and was similar to normal mice. There was no significant difference between the two AAV8 dosing groups in any test except the wire hang. In this test the performance of the lower dose treatment group declined at the later time points. Severely affected patients rapidly accumulate glycogen, and infants are often not diagnosed until they show obvious myotonia. To model the treatment of this patient population, we administered AAV8/DC190-GAA to 10-month-old Pompe mice, an age at which they display significant muscle pathology and motor function decline. Mice treated at this advanced stage of the disease showed a partial recovery of muscle function, but performance was not normalized despite high circulating levels of GAA. We are currently measuring glycogen levels and studying the histopathology of muscle and CNS tissues from the mice in these studies. In summary, we have shown that systemic gene therapy treatment is capable of preventing degenerative myopathy and preserving motor function in Pompe mice, but is less effective in animals with pre-existing pathology. I am an employee of Genzyme Corporation and hold stock in the company.
34. Phi C31 Integrase System Enhances Dystrophin Gene Expression in Skeletal Muscle of Mouse Models for Duchenne Muscular Dystrophy Carmen Bertoni,1 Sohail Jarrahian,2 Thurman M. Wheeler,1 Yining Li,1 Eric C. Olivares,2 Michele P. Calos,2 Thomas A. Rando.1 1 Neurology, Stanford University, Stanford, CA; 2Genetics, Stanford University, Stanford, CA. Duchenne muscular dystrophy (DMD) is caused by lack of dystrophin expression in skeletal muscles. 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. Plasmidbased gene therapies have been shown to be a valid approach to the treatment of a variety of disorders including DMD. Among the limitations of this technology are the restricted distribution of plasmid following direct intramuscular injection, the challenge of achieving widespread plasmid delivery by systemic injection, and the loss of extrachromosomal plasmid over time. In order to address the latter issue, which could prove to be a major limitation of therapeutic efficacy in DMD, we have studied the effects of targeted plasmid integration using a phage integrase (Phi C31) that has been shown to mediate the integration of suitably modified plasmids into the mammalian genome. Using a luciferase expression plasmid, we were able to monitor plasmid gene expression non-invasively in living mice by bioluminescence imaging (BLI). 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 fC31 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 BLIS. Shortly after delivery, the Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy