912. Accelerated Chondrogenesis in Nanofiber Scaffolds Containing BMP-2 Genetically Engineered Chondrocytes

MUSCLE AND CONNECTIVE TISSUE II with/without MCK enhancer (Me) or the human desmin promoter (Dp) with/without desmin enhancer (De). We analysed EGFP expression level of the MS-vectors with that of the MNDp in C2C12 myoblasts and myotubes after induction of cell fusion. The highest expression level was obtained with the MCKp-lentiviral vector reaching up to 25% of the MNDp level. Myoblasts from GSDII mice were transduced with either MND-GFP/GAA or Mp-GFP/ GAA lentiviral vectors at MOI 5 or 50. Transduction efficiency ranged from 25 to 69% for MNDp versus 13 to 40% for Mplentivectors. In primary myoblasts, a 14-fold higher EGFP expression level was observed for MNDp vs Mp promoter (1234 ± 243 vs 90 ± 5 MFI). A complete restoration of GAA activity was observed after transduction with MNDp-based vectors (5 to 25-fold increase vs normal cells). We demonstrated also that GAA was efficiently secreted and uptaken by GSDII myoblasts leading to an almost complete enzymatic restoration (74% activity vs normal). Only a modest increase enzymatic activity was obtained after GAA gene transfer using the Mp-based vector (11% GAA activity vs normal cells). We are currently testing a new MS lentiviral vector based on the synthetic C5-12 promoter which results will be presented at the meeting. Finally our data demonstrate that SIN-lentiviral vectors expressing human GAA can efficiently transduce GSDII myoblasts leading to a metabolic correction. These vectors form the basis of new therapeutical strategies for Pompe disease using geneticallymodified autologous muscle stem cell transplantation.

911. C-Terminal Truncated Microdystrophin Recruits Dystrobrevin and Syntrophin to the Dystrophin-Associated Glycoprotein Complex and Reduces Muscular Dystrophy in Symptomatic Utrophin/Dystrophin Double Knock-Out Mice

912. Accelerated Chondrogenesis in Nanofiber Scaffolds Containing BMP-2 Genetically Engineered Chondrocytes Robert T. Gorsline,1,3 Jin Nam,4 Prasarn Tangkawattana,2 John Lannutti,4 Alicia L. Bertone.1,2,3 1 Comparative Orthopedics Research Laboratories, Veterinary Clinical Sciences, Ohio State Univ College of Veterinary Medicine, Columbus, OH; 2Veterinary Biosciences, Ohio State Univ College of Veterinary Medicine, Columbus, OH; 3Orthopedics, Ohio State Univ College of Medicine, Columbus, OH; 4Biomedical Engineering, Ohio State Univ College of Engineering, Columbus, OH. Articular cartilage injury and erosion is a common cause of joint pain often leading to osteoarthritis and permanent morbidity. This research sought to determine if genetically engineered chondrocytes can sustain chondrogenesis in a biodegradable nanofiber scaffold. We hypothesized that chondrocytes would adhere, seed, proliferate, and produce extracellular matrix proteins typical of articular cartilage within a polycaprolactone nanofiber scaffold and this would be accelerated and more robust with hBMP-2 genetically engineered cells. Equine chondrocytes were seeded onto electrospun polycaprolactone nanofiber sheets of 150-200µm thickness and 10µm pore size and cultured in Snapwell plates across a serum gradient of 10% or 30% FBS for 2, 7, 14 and 21 days at 0.5, 1.0, and 1.5 x 106 cells/ml density. Best chondrocyte penetration into the scaffold was achieved with 1 x 106 cell seeding density and 30% serum gradient in which cells on the surface at day 2 penetrated 3/4 into the scaffold by day 7.

Yongping Yue,1 Mingju Liu,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO.

1

C-terminal truncated (∆C) microdystrophin is being developed for Duchenne muscular dystrophy (DMD) gene therapy. Encouraging results have been achieved in the mdx mouse model. Unfortunately, mdx mice do not display the same phenotype as human patients. Evaluating ∆C microdystrophin in a symptomatic model will be of significant relevance to human trial. Utrophin/ dystrophin double knock-out (u-dko) mice were developed to mold severe dystrophic changes in human patients. In this study we evaluated therapeutic effect of the ∆R4-R23/∆C microdystrophin gene (∆R4/∆C) after serotype-6 adeno-associated virus (AAV-6)mediated gene transfer in neonatal u-dko muscle. At two months after gene transfer, percentage of centrally nucleated myofiber was reduced from 89.2% to 3.4% and muscle weight was normalized. Furthermore, we have demonstrated for the first time that ∆C microdystrophin can eliminate interstitial fibrosis, macrophage infiltration and restore dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex. Interestingly neuronal nitric oxide synthase was not restored. The most impressive results were achieved in muscle force measurement. Neonatal gene therapy increased twitch and tetanic specific force. It also brought the response to eccentric contraction-induced injury to the normal range. In summary, our results suggest that the ∆R4/∆C microgene holds great promise in preventing muscular dystrophy.

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

Using these conditions, chondrocytes were cultured in triplicate either untreated, transduced with Adenoviral [Ad]-GFP, or transduced with Ad-human (h) bone morphogenetic protein (BMP)-2 for 14 days. Constructs were quantitatively evaluated for cell proliferation (DNA [ug/ml]), % viability (confocal microscopy), and matrix expression (proteoglycan (ng/ml), aggrecan (ddCT), collagen 1 (ddCT), and collagen II (ddCT). Using equine specific primers and probes, mRNA levels were expressed as ratios to 18Srna and to controls (ddCT;Fig 2). BMP-2 production and transduction efficiency (%GFP+cells) were compared. Viable cells (>90%) and high transduction efficiency (>80%) were confirmed within the biodegradable three-dimensional nanofiber scaffold using confocal and scanning electron microscopy. Accelerated and more robust expression and production of BMP-2, aggrecan and collagen II, and to a lesser extent collagen 1, persisted in the AdBMP2 treated chondrocytes within the scaffold.

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MUSCLE AND CONNECTIVE TISSUE II Use of nanofiber scaffolds to culture chondrocytes is novel and yielded a cell-supporting, biocompatible, and biodegradable tissueand genetically-engineered construct for potential use in one-step articular cartilage repair in vivo.

913. Histone Deacetylase Inhibitors Improve Gene Transfer To Mature Skeletal Muscle Nancy Larochelle,1 Jatinderpal R. Deol,1 Paul Holland,1 George Karpati,1 Josephine Nalbantoglu.1 1 Neuroimmunology, Montreal Neurological Institute, Montreal, QC, Canada. AdV-mediated gene transfer occurs inefficiently in adult skeletal muscle due to downregulation of the primary adenovirus receptor CAR during muscle maturation. We have shown previously that AdV-mediated gene transfer is enhanced in CAR transgenics, indicating that prior modulation of CAR expression results in very efficient AdV transducibility of mature skeletal muscle. We hypothesized that transient CAR expression could be achieved through activation of the transcription of the endogenous CAR gene using histone deacetylase (HDAC) inhibitors. HDACs are known to regulate gene expression via the modulation of chromatin structure by histone acetylation and deacetylation. To assess the effect of histone deacetylase inhibitors on CAR expression and adenovirus-mediated gene transfer, mouse myoblasts (C2C12) were pre-treated with the HDAC inhibitor valproic acid (VPA) and infected with AdVCMVlacZ. Quantification of ßgalactosidase activity 24 hours post-infection revealed a 5-100 fold increase in enzyme activity which depended on the multiplicity of infection (MOI). CAR expression, in cells treated with VPA, increased by a factor 3 as determined by Western blotting. VPA treatment had minimal effect on C2C12 myoblast transducibility by an Ad35 expressing lacZ, indicating that the effect is specific for the Ad5 receptor CAR. Similar treatment of primary myoblast cultures derived from a patient afflicted with Duchenne muscular dystrophy (DMD) also produced a comparable 5-300 - fold increase in ß-galactosidase activity when the VPA-treated cells were transduced with AdCMVlacZ at various MOI. To assess in vivo effects, adult normal and dystrophin-deficient (mdx) mice were treated with VPA by intraperitoneal injection (ip). Gene transfer was evaluated 7 days after intramuscular injection of AdCMVlacZ. In normal mice, no difference was seen in the number of transduced fibers and in the ß-galactosidase activity between VPA-treated animals and controls. On the other hand, a 2-fold increase was detected in mdx mice pretreated with VPA compared to non-pretreated mice. This augmentation in transduced fibers in mdx muscles correlated with an increase in CAR transcript (up to 7fold) as determined by qRT-PCR. The HDAC inhibitor valproic acid renders muscle cells more susceptible to adenovirus-mediated gene transfer. This may have future implications for muscle-directed gene therapy in muscular dystrophies.

914. Recombinant AAV Gene Delivery of Follistatin for Muscle Enhancement in Models of Muscular Dystrophy Liza Rizo,1 Chris Shilling,1 Amanda Haidet,1 Priya Umapathi Umapathi,1 Zarife Sahenk,1 Jerry R. Mendell,1 Brian K. Kaspar.1 1 Gene Therapy and Neuromuscular Research, Columbus Children’s Research Institute/The Ohio State University, Columbus, OH. Objective: To determine the efficacy of recombinant adenoassociated virus (rAAV) delivering follistatin (FS), a potent inhibitor of myostatin, to a mouse model for limb-girdle muscular dystrophy and wild-type animals. S352

Background: LGMD2D is a debilitating muscle disease of children and young adults. There is no proven treatment to delay the disease progression. Inhibition of myostatin, a negative growth modulator for muscle, can functionally improve normal or dystrophic muscle. This has been observed across species lines, through genetic manipulation, naturally occurring mutations or protein-specific antibodies. We have employed a novel gene therapy approach using a rAAV vector carrying a FS transgene. Design/Methods: Four-week-old wild-type, mdx, or alphasarcoglycan deficinet mice were injected bilaterally into the quadriceps and tibialis anterior muscles with 1x10e10 viral genomes of rAAV1FS and evaluated for muscle strength and endurance using hindlimb and forelimb grip strength as well as Rotarod. At age 120 days, gross muscle evaluation and analysis of muscle weight, fiber number, and cross-sectional area (CSA) on H&E sections was performed with the addition of a FS ELISA assay. Results: We found increased serum FS levels accompanied by a local and remote increase in muscle mass in treated animals (p<0.05). Muscle hypertrophy was observed in rAAV-FS mice versus controls. Motor function tests showed statistically significant improvements in muscle strength in FS-treated mice. Conclusions/Relevance: This data supports a therapeutic effect in muscular dystrophy by myostatin inhibition. This approach represents a clinically applicable gene therapy method to enhance muscle mass and function in muscular dystrophy with potential for treatment of other muscle diseases. No toxicity was encountered. The FS transgene could also be used to complement gene replacement therapy. For example, adding muscle mass in patients with more advanced dystrophies could improve their candidacy for gene replacement therapy.

915. High and Low Affinity Consensus Binding Sequences to Extracellular α7 Integrin X2B Subunit Detected Using Phage Display Biopanning Daniel P. Reay,1 Pattarana Sae-chew,1 Paula R. Clemens.1,2 Neurology, University of Pittsburgh, Pittsburgh, PA; 2Neurology Service, Department of Veterans Affairs Medical Center, Pittsburgh, PA. 1

Multiple biopanning strategies have been developed to uncover ligands that bind to specific cellular receptors such as panning on immobilized targets or on live cells. This study utilized a cell-based system in which MCF7 breast carcinoma cells stably transfected with the α7 integrin X2B subunit gene (MCF7α7(+)) were used for positive selection, while non-transfected MCF7 cells were used for negative selection. We chose α7 integrin, a cell surface receptor whose expression is largely restricted to skeletal muscle and is upregulated in dystrophic muscle, as our biopanning receptor to discover targeting ligands for muscle gene delivery. The biopanning strategy developed for this project began with a single library of phage representing approximately 1x109 possible combinations of 7 amino acids. For the first round of biopanning, we incubated the original library with MCF7α7(+) cells for positive selection, removed any non-binding phage and collected the bound phage in 2 separate fractions. A low affinity fraction was collected by low pH wash and then neutralized, while a high affinity fraction was recovered by cell collection, subsequent freezing and thawing to lyse the cells, and removal of cellular debris by centrifugation. Once the positive selection was completed, we immediately performed negative selection using each fraction (low and high affinity) separately on MCF7 cells. After the last of 6 rounds of biopanning for each phage fraction, with each round containing a positive and negative selection, the un-amplified eluate was titered to limiting dilution and individual phage clones were picked and amplified separately. Purified clones were analyzed first by an ELISA-based binding assay developed Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy