185 AAV micro dystrophin therapy alleviates stress induced cardiac death but does not reduce myocardial fibrosis in >21 m old mdx mice

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[.]

Molecular Therapy Volume 20, Supplement 1, May 2012

MUSCULO-SKELETAL GENE & CELL THERAPY I

peritoneal cavity Nine times transplantations, two millions of

dko-MSC 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 myofi ber 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 Identifi cation 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; 2 Laboratoire 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

anti-myostatin 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 signifi cant 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

31-71 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

suffi cient 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

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 signifi cantly to its binding affi nity, 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 myofi ber 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 signifi cantly 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 fi rst 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-defi cient muscle disease We recently demonstrated amelioration of dystrophic heart disease in 16 to 20-m-old dystrophin null mdx mice using adeno-associated 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 fi brosis and heart function were examined

Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy

S74

CANCER – IMMUNOTHERAPY I

at 1.7 ± 0.2 months after gene therapy Effi cient micro-dystrophin

expression was observed in the myocardium in treated mice However,

myocardial fi brosis 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 fi rst time the potential benefi ts and limitations of AAV

micro-dystrophin therapy in end-stage Duchenne cardiomyopathy

Our fi ndings 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

1 Department of Molecular Therapy, National Institute of

Neuroscience, National Center of Neurology and Psychiatry,

Kodaira, Tokyo, Japan; 2 Department of Pediatrics, School of

Medicine, Tokyo Women’s Medical University, Shinjuku, Tokyo,

Japan; 3 Department 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 diffi cult, therefore utilizing of fi broblasts

is considered We have previously reported the FACS-utilizing MyoD

transduction of fi broblasts, 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 fi broblasts,

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 fi ndings suggest

that the expression of myogenic marker protein on MyoD-transduced

fi broblasts would be benefi cial to effi ciently 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 fi broblasts 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 Post-Operative 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

1 Mary Crowley Cancer Research Centers, Dallas; 2 Medical City HCA, Dallas; 3 Texas Oncology, P.A., Dallas; 4 Gradalis, Inc., Dallas; 5 WLS 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 suffi cient safety data to proceed to Phase II effi cacy studies This decision was bolstered by the correlation of survival with induced immune response (IFNgamma ELISPOT) in serially monitored patients at ≥ 4 months (p=0.025) Specifi cally, 6 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 2 patients at dose level 1 x 107 and 4 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) Effi cacy of the pbi-shRNA™ FURIN vaccine component was confi rmed 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 modifi ed 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, 3 then q 4 weeks with mFOLFOX starting Week 4 then continuing q 2 weeks x 6 months

A minimum harvest aliquot to produce 5 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 infi ltrating 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

1 Molecular Biology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA; 2 Department of Surgery, Surgical Oncology, UCLA, Los Angeles, CA; 3 Department 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