- Email: [email protected]
182. A Novel Cell Therapy for Muscular Dystrophy by Bone Marrow Stromal Cell: Mesenchymal Stem Cell Derived from Bone Marrow Can Affect Skeletal Muscle Regeneration
MUSCULO-SKELETAL GENE & CELL THERAPY I utrn-/- mice to determine if there is a similar improvement in muscle physiology and investigating the mechanism that is involved in the protection from eccentric contraction induced injury.
179. Systemic Delivery of nNOS-Recruiting Mini-Dystrophin AAV Vectors Ameliorated Muscular Dystrophy in a Mouse DMD Model
Yadong Zhang,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO.
1
The nNOS-recruiting mini-dystrophin gene is a promising candidate gene for Duchenne muscular dystrophy (DMD) gene therapy. We recently developed a set of dual AAV vectors (YZ27/ YZ22) to express the nNOS-recruiting minigene. Local injection yielded robust transduction and sarcolemmal nNOS localization (Zhang & Duan Hum Gene Ther 23:98-103, 2012). Here, we examined the therapeutic efficacy of this dual vector set following systemic gene transfer. We packaged YZ27/YZ22 in AAV-9 and administered to 3-week-old dystrophin-deficient mdx mice via tail vein injection. Four months later we examined minigene expression, histopathology and muscle force. Immunohistochemistry and Western blot revealed broad mini-dystrophin expression in skeletal muscle. Inflammation and central nucleation were reduced in treated mice. Physiology assay showed significant improvement of specific muscle force in both anterior tibialis and extensor digitorum longus muscles. These data have further demonstrated therapeutic efficacy of the nNOS recruiting mini-dystrophin dual AAV vectors. Further evaluation of this vector set in large animal models may set the foundation for future clinical trial (Supported by NIH and MDA).
180. Adeno Associated Virus (AAV) Mediated Follistatin Gene Transfer Toxicology Studies in Preparation of Phase I/II Clinical Trial
Janaiah Kota,1 Christopher Shilling,1 Chrystal Montgomery,1 Sarah Lewis,1 Adam Bevan,1 Kim Shontz,1 Yuuki Kaminoh,1 Kristin Heller,1 Xiomara Rosales,1 Laurence Viollet,1 Kevin Flanigan,1 K. Reed Clark,1 Brian Kaspar,1 Zarife Sahenk,1 Jerry Mendell.1 1 Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital, Columbus, OH. Inhibition of myostatin signaling is a therapeutic strategy to improve muscle size and strength for muscle disease. Follistatin has been demonstrated as a potent myostatin antagonist. In our previous pre-clinical work in both rodents and non-human primates, intramuscular (IM) administration of an alternatively spliced follistatin isoform, FS344, delivered using AAV serotype1 effectively improved muscle mass and strength with no off target effects. To establish the safety of rAAV1.CMV.follistatin344 for clinical trial use, a dose-ranging toxicology study was carried out in mice including a dose 10-fold greater than that planned for a phase I/II gene therapy clinical trial. C57BL/6 mice were randomized to three experimental arms delivering rAAV1.CMV.follistatin344 bilaterally to quadriceps muscle by IM injection. Dose ranging corresponded to the highest clinical dose (2x1012 vg/kg) or 10-fold higher (2x1013 vg/kg) compared to vector diluent. Animals were necropsied at 6, 12, 24 and 36 weeks (10 animals/timepoint) post injection (PI). Outcomes included: Body weight, hematology, chemistry panels, reproductive hormones, immune studies, viral vector biodistribution and histopathology of 24 organs from each animal. No treatment related adverse clinical signs were observed throughout the study. None of the animals showed abnormalities in hematology, clinical chemistry parameters and reproductive hormones and viral DNA dissipated in non-injected tissues by 24 weeks. At 6 weeks PI, in high dose animals, scattered focal infiltrates of inflammatory cells were observed in the injected muscle consistent with T-cell immunity S72
to AAV1 as identified by IFN-g ELISpot, but no other pathology was observed in any of the organs. Increase in quadriceps muscle mass was observed at all-time points that correlated with myofiber hypertrophy and muscle follistatin expression. Mean fiber diameter increased and remained persistent from 6 weeks (63.3 ±2.1 μm) to 36 weeks (65.9 ± 2.1 μm) PI compared to controls (46.6 ± 5.1μm). The density of PaX7 positive nuclei increased rapidly at 6 weeks, prior to the occurrence of hypertrophic-multinucleated muscle fibers at 12 weeks. No circulating antibody to follistatin was observed. In conclusion, this study shows rAAV1.CMV.follistatin344 is safe and well tolerated at the proposed clinical doses and causes satellite cell proliferation and significant muscle hypertrophy by fusion with differentiated satellite cells. Early transient inflammation in muscle is consistent with an immune response to AAV1. These results support this approach for clinical trial use.
181. Abbreviated Dystrophins Restore the Passive Properties of the Extensor Digitorum Longus Muscle in Dystrophin-Null Mice
Chady H. Hakim,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO.
1
Muscle and joint stiffness is a major clinical feature in Duchenne muscular dystrophy (DMD) patients. DMD is one of the most common lethal inherited muscle wasting diseases in childhood and it is caused by the loss of the dystrophin protein. We recently showed that the extensor digitorum longus (EDL) muscle of mdx mice (a DMD mouse model) exhibits disease-associated muscle stiffness. Abbreviated forms of the dystrophin gene (micro and mini-dystrophin) are the leading candidate genes for DMD gene therapy. Unfortunately, it has never been clear whether these abbreviated genes can mitigate muscle stiffness. To address this critical question, we examined the passive properties (elastic and viscous properties) of the EDL muscle in transgenic mdx mice that express either a representative minigene (ΔH2-R15) or one of the two most promising microgenes (ΔR2-15/ΔR18-23/ΔC or ΔR4-23/ ΔC). The passive properties were measured in 6, 14, and 20-month-old transgenic mice and the results were compared to those of age and sex-matched normal and mdx mice. Surprisingly, despite significant truncation of the gene, the elastic and viscous properties were completely normalized in every strain of transgenic mice we examined. Our results demonstrate for the first time that abbreviated dystrophin genes can effectively treat muscle stiffness in a DMD model. Our findings provide important support to further develop mini-/micro-dystrophin gene therapy.
182. A Novel Cell Therapy for Muscular Dystrophy by Bone Marrow Stromal Cell: Mesenchymal Stem Cell Derived from Bone Marrow Can Affect Skeletal Muscle Regeneration
Yasushi Maeda,1 Asuka Koga,1 Masatoshi Ishizaki,2 Tomohiro Suga.1 1 Neurology, Kumamoto University Faculty of Life Sciences, Kumamoto, Japan; 2Neurology, Kumamoto Saishunso National Hospital, Koshi, Kumamoto, Japan.
Bone marrow stromal cells are stem cells differentiating in various mesenchymal tissues: bone, fat, skeletal muscle and so on. These cells are called MSC and widely noticed as an ideal cell for regenerative medicine. Its immunomodulatory competence becomes evident as well. MSC having these multi biological properties is very interesting in therapeutic application for various diseases. We hypothesized that MSC may affect the regeneration of mesenchymal tissue like skeletal muscle. In order to prove this hypothesis, we repetitively transplanted MSC (dko-MSC) from a muscular disease model mouse, dystrophin / utrophin double knockout mouse (D/U dko), into D/U dko mouse’s Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
MUSCULO-SKELETAL GENE & CELL THERAPY I peritoneal cavity. Nine times transplantations, two millions of dkoMSC in each transplantation, were carried out by 8-week-old. The transplantation obviously improved the body build, skeletal structure and locomotor activity.
Histologically, the transplantation made myofiber hypertrophic comparing to the wild-type mouse’s myofiber. Surprisingly, transplantation drastically prolonged life span.
We can prove that MSC modifes the skeletal muscle regeneration. Although the mechanism is still unknown, autologous MSC transplantation to patient suffering from muscular dystrophy will become a novel therapy. More importantly, this cell therapy may be applied not only to muscular dystrophy but also common myopathy.
183. Identification of Decorin Derived Peptides with a Zn2+ Dependent Anti-Myostatin Activity
Simon Guiraud,1,2 Laetitia van Wittenberghe,1 Christophe Georger,1 Daniel Scherman,2 Antoine Kichler.1,2 1 Genethon, Evry, France; 2Laboratoire de Pharmacologie Chimique et Génétique - CNRS UMR8151, U1022 Inserm, Université Paris Descartes, Chimie Paristech, Paris, France. Decorin is a member of the small leucine-rich proteoglycan (SLRP) family and it is a component of the extracellular matrix. Decorin was previously shown to bind different molecules, including myostatin, in a zinc-dependent manner. Here, we investigated in detail the antimyostatin activity of decorin and fragments thereof. We show that this protein displays in vitro anti-myostatin activities with an IC50 of 2.3.10-8M. After intramuscular injection of decorin in dystrophic mdx and γ-sarcoglycan-/- mice, we observed a significant increase of the muscle mass and this effect was maximal 18 days after administration. Further, we show that the myostatin-binding site is located in the N-terminal domain of decorin. In fact, a peptide encompassing the 3171 sequence retains full myostatin binding capacity and intramuscular injection of the peptide induces muscle hypertrophy. The evaluation of three additional peptides suggests a crucial role of the four cysteines within the conserved CX3CXCX6C motif of class I of the SLRPs. Altogether, our results show that the N-terminal domain of decorin is sufficient for the binding to myostatin and they underscore the crucial role for this interaction of zinc and the cysteine cluster. We are now working on gene therapy approaches based on the use of expression vectors encoding the decorin protein or peptide. Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
184. Follistatin N-Terminal Mutation Diminishes Muscle Growth Effects In Vivo
Chunping Qiao,1 Hui Zheng,1 Ruhang Tang,1 Chihsien Wang,1 Jianbin Li,1 Tiffany Chou,1 Juan Li,1 Xiao Xiao.1 1 Department of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, Chapel Hill, NC. Follistatin has been emerged as a robust antagonist of myostatin, protein or gene delivery of Follistatin to promote muscle growth is considered a promising strategy for the treatment of muscle wasting diseases such as muscular dystrophies. Follistatin is a multi-domain protein consisting of an N-terminal domain (ND), three subsequent domains (FSD1-3) and a C-terminal tail. While it is widely accepted that FSD1 and FSD2 contribute significantly to its binding affinity, the function of N-terminal domain of follistatin is not clearly understood. The purpose of this study is to investigate the role of N-terminal domain of follistatin, and the understanding of it will help delineate therapeutic mechanism of follistatin. The N-terminus mutant dog follistatin cDNA (GenBank XM_536475), normal dog follistatin cDNA (GenBank DD116838), and normal mouse follistatin cDNA were cloned into adeno-associated viral (AAV) vector plasmids driven by ubiquitous CAG (cytomegalovirus early enhancer and chick beta-actin) promoter, and their functions were evaluated both in vitro and in vivo. The in vitro TGF-beta1-responsive luciferase reporter assay indicated that the N-terminus mutant dog follistatin did not inhibit the luciferase activity induced by activin, a strong follistatin binding partner, while normal follistatins indeed significantly blocked its activity. For in vivo study, AAV9 vector containing different versions of follistatin gene were delivered into normal mice via systemic delivery, and the general growth and activities of treated mice were compared. Overexpression of normal mouse or dog follistatin was robust in increasing body weight, muscle mass and myofiber sizes, whereas overexpression of N-terminus mutant dog follistatin had no effect on them. Interestingly, motor function data revealed that overexpression of normal versions of follistatin could increase grip force, rotor-rod climbing time, but could not increase the treadmill running distance of the treated mice. On the other hand, overexpression of N-terminus mutant dog follistatin could significantly increase the treadmill running distance of the treated mice, although it had no effect on grip force and rotor-rod climbing ability. Thus, our study provided the first in vivo evidence of the function of N-terminal domain of follistatin, shedding light on future new molecular design for follistatin gene therapy for muscle wasting diseases.
185. AAV Micro-Dystrophin Therapy Alleviates Stress-Induced Cardiac Death but Does Not Reduce Myocardial Fibrosis in >21-m-Old mdx Mice
Brian Bostick,1 Jin-Hong Shin,1 Yongping Yue,1 Yi Lai,1 Nalinda B. Wasala,1 Dongsheng Duan.1 1 Molecular Microbiology & Immunology, School of Medicine, The University of Missouri, Columbia, MO. The incidence of heart disease increases markedly with age in Duchenne muscular dystrophy (DMD), a dystrophin-deficient muscle disease. We recently demonstrated amelioration of dystrophic heart disease in 16 to 20-m-old dystrophin null mdx mice using adenoassociated virus (AAV) mediated micro-dystrophin gene therapy. Like DMD patients, the severity of heart disease worsens profoundly when mdx mice reach beyond 21 months of age. To more rigorously test micro-dystrophin therapy, here we treated mdx mice that were between 21.2 to 22.7-m-old (average, 22.1 ± 0.2 months; N=8). The ΔR4-23/ΔC micro-dystrophin gene was packaged in AAV-9. 5 x 1012 viral genome particles/mouse were delivered via the tail vein. AAV transduction, myocardial fibrosis and heart function were examined S73
179. Systemic Delivery of nNOS-Recruiting Mini-Dystrophin AAV Vectors Ameliorated Muscular Dystrophy in a Mouse DMD Model
Yadong Zhang,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO.
1
The nNOS-recruiting mini-dystrophin gene is a promising candidate gene for Duchenne muscular dystrophy (DMD) gene therapy. We recently developed a set of dual AAV vectors (YZ27/ YZ22) to express the nNOS-recruiting minigene. Local injection yielded robust transduction and sarcolemmal nNOS localization (Zhang & Duan Hum Gene Ther 23:98-103, 2012). Here, we examined the therapeutic efficacy of this dual vector set following systemic gene transfer. We packaged YZ27/YZ22 in AAV-9 and administered to 3-week-old dystrophin-deficient mdx mice via tail vein injection. Four months later we examined minigene expression, histopathology and muscle force. Immunohistochemistry and Western blot revealed broad mini-dystrophin expression in skeletal muscle. Inflammation and central nucleation were reduced in treated mice. Physiology assay showed significant improvement of specific muscle force in both anterior tibialis and extensor digitorum longus muscles. These data have further demonstrated therapeutic efficacy of the nNOS recruiting mini-dystrophin dual AAV vectors. Further evaluation of this vector set in large animal models may set the foundation for future clinical trial (Supported by NIH and MDA).
180. Adeno Associated Virus (AAV) Mediated Follistatin Gene Transfer Toxicology Studies in Preparation of Phase I/II Clinical Trial
Janaiah Kota,1 Christopher Shilling,1 Chrystal Montgomery,1 Sarah Lewis,1 Adam Bevan,1 Kim Shontz,1 Yuuki Kaminoh,1 Kristin Heller,1 Xiomara Rosales,1 Laurence Viollet,1 Kevin Flanigan,1 K. Reed Clark,1 Brian Kaspar,1 Zarife Sahenk,1 Jerry Mendell.1 1 Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital, Columbus, OH. Inhibition of myostatin signaling is a therapeutic strategy to improve muscle size and strength for muscle disease. Follistatin has been demonstrated as a potent myostatin antagonist. In our previous pre-clinical work in both rodents and non-human primates, intramuscular (IM) administration of an alternatively spliced follistatin isoform, FS344, delivered using AAV serotype1 effectively improved muscle mass and strength with no off target effects. To establish the safety of rAAV1.CMV.follistatin344 for clinical trial use, a dose-ranging toxicology study was carried out in mice including a dose 10-fold greater than that planned for a phase I/II gene therapy clinical trial. C57BL/6 mice were randomized to three experimental arms delivering rAAV1.CMV.follistatin344 bilaterally to quadriceps muscle by IM injection. Dose ranging corresponded to the highest clinical dose (2x1012 vg/kg) or 10-fold higher (2x1013 vg/kg) compared to vector diluent. Animals were necropsied at 6, 12, 24 and 36 weeks (10 animals/timepoint) post injection (PI). Outcomes included: Body weight, hematology, chemistry panels, reproductive hormones, immune studies, viral vector biodistribution and histopathology of 24 organs from each animal. No treatment related adverse clinical signs were observed throughout the study. None of the animals showed abnormalities in hematology, clinical chemistry parameters and reproductive hormones and viral DNA dissipated in non-injected tissues by 24 weeks. At 6 weeks PI, in high dose animals, scattered focal infiltrates of inflammatory cells were observed in the injected muscle consistent with T-cell immunity S72
to AAV1 as identified by IFN-g ELISpot, but no other pathology was observed in any of the organs. Increase in quadriceps muscle mass was observed at all-time points that correlated with myofiber hypertrophy and muscle follistatin expression. Mean fiber diameter increased and remained persistent from 6 weeks (63.3 ±2.1 μm) to 36 weeks (65.9 ± 2.1 μm) PI compared to controls (46.6 ± 5.1μm). The density of PaX7 positive nuclei increased rapidly at 6 weeks, prior to the occurrence of hypertrophic-multinucleated muscle fibers at 12 weeks. No circulating antibody to follistatin was observed. In conclusion, this study shows rAAV1.CMV.follistatin344 is safe and well tolerated at the proposed clinical doses and causes satellite cell proliferation and significant muscle hypertrophy by fusion with differentiated satellite cells. Early transient inflammation in muscle is consistent with an immune response to AAV1. These results support this approach for clinical trial use.
181. Abbreviated Dystrophins Restore the Passive Properties of the Extensor Digitorum Longus Muscle in Dystrophin-Null Mice
Chady H. Hakim,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO.
1
Muscle and joint stiffness is a major clinical feature in Duchenne muscular dystrophy (DMD) patients. DMD is one of the most common lethal inherited muscle wasting diseases in childhood and it is caused by the loss of the dystrophin protein. We recently showed that the extensor digitorum longus (EDL) muscle of mdx mice (a DMD mouse model) exhibits disease-associated muscle stiffness. Abbreviated forms of the dystrophin gene (micro and mini-dystrophin) are the leading candidate genes for DMD gene therapy. Unfortunately, it has never been clear whether these abbreviated genes can mitigate muscle stiffness. To address this critical question, we examined the passive properties (elastic and viscous properties) of the EDL muscle in transgenic mdx mice that express either a representative minigene (ΔH2-R15) or one of the two most promising microgenes (ΔR2-15/ΔR18-23/ΔC or ΔR4-23/ ΔC). The passive properties were measured in 6, 14, and 20-month-old transgenic mice and the results were compared to those of age and sex-matched normal and mdx mice. Surprisingly, despite significant truncation of the gene, the elastic and viscous properties were completely normalized in every strain of transgenic mice we examined. Our results demonstrate for the first time that abbreviated dystrophin genes can effectively treat muscle stiffness in a DMD model. Our findings provide important support to further develop mini-/micro-dystrophin gene therapy.
182. A Novel Cell Therapy for Muscular Dystrophy by Bone Marrow Stromal Cell: Mesenchymal Stem Cell Derived from Bone Marrow Can Affect Skeletal Muscle Regeneration
Yasushi Maeda,1 Asuka Koga,1 Masatoshi Ishizaki,2 Tomohiro Suga.1 1 Neurology, Kumamoto University Faculty of Life Sciences, Kumamoto, Japan; 2Neurology, Kumamoto Saishunso National Hospital, Koshi, Kumamoto, Japan.
Bone marrow stromal cells are stem cells differentiating in various mesenchymal tissues: bone, fat, skeletal muscle and so on. These cells are called MSC and widely noticed as an ideal cell for regenerative medicine. Its immunomodulatory competence becomes evident as well. MSC having these multi biological properties is very interesting in therapeutic application for various diseases. We hypothesized that MSC may affect the regeneration of mesenchymal tissue like skeletal muscle. In order to prove this hypothesis, we repetitively transplanted MSC (dko-MSC) from a muscular disease model mouse, dystrophin / utrophin double knockout mouse (D/U dko), into D/U dko mouse’s Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
MUSCULO-SKELETAL GENE & CELL THERAPY I peritoneal cavity. Nine times transplantations, two millions of dkoMSC in each transplantation, were carried out by 8-week-old. The transplantation obviously improved the body build, skeletal structure and locomotor activity.
Histologically, the transplantation made myofiber hypertrophic comparing to the wild-type mouse’s myofiber. Surprisingly, transplantation drastically prolonged life span.
We can prove that MSC modifes the skeletal muscle regeneration. Although the mechanism is still unknown, autologous MSC transplantation to patient suffering from muscular dystrophy will become a novel therapy. More importantly, this cell therapy may be applied not only to muscular dystrophy but also common myopathy.
183. Identification of Decorin Derived Peptides with a Zn2+ Dependent Anti-Myostatin Activity
Simon Guiraud,1,2 Laetitia van Wittenberghe,1 Christophe Georger,1 Daniel Scherman,2 Antoine Kichler.1,2 1 Genethon, Evry, France; 2Laboratoire de Pharmacologie Chimique et Génétique - CNRS UMR8151, U1022 Inserm, Université Paris Descartes, Chimie Paristech, Paris, France. Decorin is a member of the small leucine-rich proteoglycan (SLRP) family and it is a component of the extracellular matrix. Decorin was previously shown to bind different molecules, including myostatin, in a zinc-dependent manner. Here, we investigated in detail the antimyostatin activity of decorin and fragments thereof. We show that this protein displays in vitro anti-myostatin activities with an IC50 of 2.3.10-8M. After intramuscular injection of decorin in dystrophic mdx and γ-sarcoglycan-/- mice, we observed a significant increase of the muscle mass and this effect was maximal 18 days after administration. Further, we show that the myostatin-binding site is located in the N-terminal domain of decorin. In fact, a peptide encompassing the 3171 sequence retains full myostatin binding capacity and intramuscular injection of the peptide induces muscle hypertrophy. The evaluation of three additional peptides suggests a crucial role of the four cysteines within the conserved CX3CXCX6C motif of class I of the SLRPs. Altogether, our results show that the N-terminal domain of decorin is sufficient for the binding to myostatin and they underscore the crucial role for this interaction of zinc and the cysteine cluster. We are now working on gene therapy approaches based on the use of expression vectors encoding the decorin protein or peptide. Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
184. Follistatin N-Terminal Mutation Diminishes Muscle Growth Effects In Vivo
Chunping Qiao,1 Hui Zheng,1 Ruhang Tang,1 Chihsien Wang,1 Jianbin Li,1 Tiffany Chou,1 Juan Li,1 Xiao Xiao.1 1 Department of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, Chapel Hill, NC. Follistatin has been emerged as a robust antagonist of myostatin, protein or gene delivery of Follistatin to promote muscle growth is considered a promising strategy for the treatment of muscle wasting diseases such as muscular dystrophies. Follistatin is a multi-domain protein consisting of an N-terminal domain (ND), three subsequent domains (FSD1-3) and a C-terminal tail. While it is widely accepted that FSD1 and FSD2 contribute significantly to its binding affinity, the function of N-terminal domain of follistatin is not clearly understood. The purpose of this study is to investigate the role of N-terminal domain of follistatin, and the understanding of it will help delineate therapeutic mechanism of follistatin. The N-terminus mutant dog follistatin cDNA (GenBank XM_536475), normal dog follistatin cDNA (GenBank DD116838), and normal mouse follistatin cDNA were cloned into adeno-associated viral (AAV) vector plasmids driven by ubiquitous CAG (cytomegalovirus early enhancer and chick beta-actin) promoter, and their functions were evaluated both in vitro and in vivo. The in vitro TGF-beta1-responsive luciferase reporter assay indicated that the N-terminus mutant dog follistatin did not inhibit the luciferase activity induced by activin, a strong follistatin binding partner, while normal follistatins indeed significantly blocked its activity. For in vivo study, AAV9 vector containing different versions of follistatin gene were delivered into normal mice via systemic delivery, and the general growth and activities of treated mice were compared. Overexpression of normal mouse or dog follistatin was robust in increasing body weight, muscle mass and myofiber sizes, whereas overexpression of N-terminus mutant dog follistatin had no effect on them. Interestingly, motor function data revealed that overexpression of normal versions of follistatin could increase grip force, rotor-rod climbing time, but could not increase the treadmill running distance of the treated mice. On the other hand, overexpression of N-terminus mutant dog follistatin could significantly increase the treadmill running distance of the treated mice, although it had no effect on grip force and rotor-rod climbing ability. Thus, our study provided the first in vivo evidence of the function of N-terminal domain of follistatin, shedding light on future new molecular design for follistatin gene therapy for muscle wasting diseases.
185. AAV Micro-Dystrophin Therapy Alleviates Stress-Induced Cardiac Death but Does Not Reduce Myocardial Fibrosis in >21-m-Old mdx Mice
Brian Bostick,1 Jin-Hong Shin,1 Yongping Yue,1 Yi Lai,1 Nalinda B. Wasala,1 Dongsheng Duan.1 1 Molecular Microbiology & Immunology, School of Medicine, The University of Missouri, Columbia, MO. The incidence of heart disease increases markedly with age in Duchenne muscular dystrophy (DMD), a dystrophin-deficient muscle disease. We recently demonstrated amelioration of dystrophic heart disease in 16 to 20-m-old dystrophin null mdx mice using adenoassociated virus (AAV) mediated micro-dystrophin gene therapy. Like DMD patients, the severity of heart disease worsens profoundly when mdx mice reach beyond 21 months of age. To more rigorously test micro-dystrophin therapy, here we treated mdx mice that were between 21.2 to 22.7-m-old (average, 22.1 ± 0.2 months; N=8). The ΔR4-23/ΔC micro-dystrophin gene was packaged in AAV-9. 5 x 1012 viral genome particles/mouse were delivered via the tail vein. AAV transduction, myocardial fibrosis and heart function were examined S73