185 AAV Micro Dystrophin Therapy Alleviates Stress Induced Cardiac Death but Does Not Reduce Myocardial Fibrosis in >21 m Old mdx Mice Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © T[.]
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 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 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 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 x 1012 viral genome particles/mouse were delivered via the tail vein AAV transduction, myocardial fibrosis and heart function were examined S73 CANCER – IMMUNOTHERAPY I at 1.7 ± 0.2 months after gene therapy Efficient micro-dystrophin expression was observed in the myocardium in treated mice However, myocardial fibrosis was not mitigated Most hemodynamic parameters were not improved either Interestingly, treatment improved PR interval and QRS duration Further, treated mice became more resistant to dobutamine-induced cardiac death In summary, we have revealed for the first time the potential benefits and limitations of AAV micro-dystrophin therapy in end-stage Duchenne cardiomyopathy Our findings have important implications on the further development of AAV micro-dystrophin gene therapy 186 Myogenic Transduction and Cell Surface Marker Selection of DMD Fibroblasts Enable Stable Dystrophin mRNA Expression for Exon Skipping Assay Takashi Saito,1,2 Tetsuya Nagata,1 Yoshitsugu Aoki,1 Jun Tanihata,1 Satoru Masuda,1 Toshifumi Yokota,3 Yuko Shimizu Motohashi,1 Shin’ichi Takeda.1 Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan; 2Department of Pediatrics, School of Medicine, Tokyo Women’s Medical University, Shinjuku, Tokyo, Japan; 3Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada Duchenne Muscular Dystrophy (DMD) is a lethal, X-linked muscular disease, due to the loss of dystrophin protein Antisense oligonucleotide (AON) mediated exon skipping is one of promising approaches to cure this disease and phase II-III clinical trials of exon 51 skipping are ongoing Preliminary but successful results of these trials are indicating that other exons will be targeted in subsequent trials To develop effective AON for each DMD case, assays using patient-derived cells are valuable In some cases, obtaining myoblasts from patients is difficult, therefore utilizing of fibroblasts is considered We have previously reported the FACS-utilizing MyoD transduction of fibroblasts, and it was practical for exon skipping assay (PLoS One 2010 Aug 18;5(8):e12239.) Nevertheless, in our current assay system, a long-term differentiation culture is required for enough dystrophin mRNA expression To address this challenge, we evaluated an availability of several myogenic surface markers CD56 is known as one of the myogenic markers expressed on a surface of primary myoblasts We found that the MyoD-transduced fibroblasts, which were initially negative with CD56 by FACS analysis, gradually increased its positive population, and these cells expressed much dystrophin mRNA than negative population These findings suggest that the expression of myogenic marker protein on MyoD-transduced fibroblasts would be beneficial to efficiently select the cells expressing dystrophin mRNA and shorten the period required for enough dystrophin mRNA expression We would report the possibility that a stable dystrophin mRNA expression of DMD fibroblasts required for exon skipping assay might be achieved through a combination of MyoD transduction and myogenic surface marker Cancer – Immunotherapy I 187 Randomized Phase II Trial of PostOperative Adjuvant Chemotherapy ±FANG™ Autologous Tumor Cell Vaccine in Colorectal Carcinoma with Liver Metastases Joseph Kuhn,5 Neil Senzer,1,2,3,4 Minal Barve,3 Padmasini Kumar,4 Donald D Rao,4 Gladice Wallraven,4 Beena O Pappen,4 Phillip B Maples,4 John Nemunaitis.1,2,3,4 Mary Crowley Cancer Research Centers, Dallas; 2Medical City HCA, Dallas; 3Texas Oncology, P.A., Dallas; 4Gradalis, Inc., Dallas; 5WLS Surgical Associates, P.A., Dallas The Phase I trial of FANG™ vaccine (comprised of irradiated autologous tumor cells transduced with plasmid encoding GMCSF and the RNA interference bi-shRNA FURIN; see Senzer et al., Mol Ther 2011) involving 31 previously treated solid tumor patients provided sufficient safety data to proceed to Phase II efficacy studies This decision was bolstered by the correlation of survival with induced immune response (IFNgamma ELISPOT) in serially monitored patients at ≥ months (p=0.025) Specifically, metastatic refractory colon cancer patients in the Phase I trial, who having failed standard therapy, had an expected survival of ∼6 months, received a combined total of 24 vaccinations (i.e patients at dose level x 107 and patients at 2.5 x 107 cells / injection; data combined as there is no dose related difference in endpoint) These patients currently demonstrate median survivals of 358 days (554, 515+, 413, 303, 185, and 167 days each) Efficacy of the pbi-shRNA™ FURIN vaccine component was confirmed by marked knockdown of the furin proprotein convertase activated immunosuppressive proteins TGFβ1 and b2; 87% and 92% respectively) Based on these results, the Phase II trial (CL-PTL 114, BB-IND 14205) was activated following FDA, RAC, IBC and IRB acceptance This is a randomized Phase II study of integrated adjuvant modified FOLFOX6 (mFOLFOX6) and intradermal autologous FANG™ cancer vaccine (1.0 x 107 cells/injection; maximum of 12 vaccinations) versus adjuvant mFOLFOX6 and placebo in patients with colorectal carcinoma with either synchronous or metachronous liver metastases (CLM ± pulmonary metastases) following resection ± ablation with curative intent Eight weeks following resection ± ablation, patients will receive FANG™ Weeks 1, then q weeks with mFOLFOX starting Week then continuing q weeks x months A minimum harvest aliquot to produce monthly injections will be required for entry into the study These patients will be managed in an outpatient setting Hematologic function, liver enzymes, renal function and electrolytes will be monitored weekly Immune function analyses including assessment of tumor infiltrating lymphocytes (TIL) and ELISPOT analysis of cytotoxic T cell function to autologous tumor antigens will be monitored at baseline (TIL + ELISPOT) and thereafter ELISPOT analysis at Months 2, 4, 6, 9, end of treatment, and at response follow up visits Preliminary data will be presented at the meeting 188 Hematopoietic Stem Cell Immunotherapy for Melanoma Eric H Gschweng,1 Michael L Kaufman,1 Roger P Hollis,1 Koya C Richard,2 Ribas Antoni,3 Donald B Kohn.1 Molecular Biology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA; 2Department of Surgery, Surgical Oncology, UCLA, Los Angeles, CA; 3Department of Medicine, Hematology/ Oncology, UCLA, Los Angeles, CA Melanoma is the deadliest form of skin cancer, and the primary treatment option of surgical removal becomes infeasible with widespread metastasis Immunotherapy involves redirecting the immune system with transgenic T-cell receptors (TCR), and offers an attractive strategy to combat this disease While transduction S74 Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... – IMMUNOTHERAPY I at 1.7 ± 0.2 months after gene therapy Efficient micro- dystrophin expression was observed in the myocardium in treated mice However, myocardial fibrosis was not mitigated Most... hemodynamic parameters were not improved either Interestingly, treatment improved PR interval and QRS duration Further, treated mice became more resistant to dobutamine -induced cardiac death In. .. deadliest form of skin cancer, and the primary treatment option of surgical removal becomes infeasible with widespread metastasis Immunotherapy involves redirecting the immune system with transgenic