140. Gait in BioTO-2 and Bio14.6 Dystrophic Hamsters

virus (rAAV) vectors expressing the human o-sarcoglycan cDNA under the control of a muscle specific promoter. Efficient and sustained transgene expression with correct sarcolemmal localization and without evident toxicity was obtained after intra-arterial injection into the both hindlimbs of a LGMD2D murine model. Transgene expression resulted in restoration of the sarcoglycan complex, histological improvement, membrane stabilization with full rescue of the contractile force deficits and stretch sensibility that led to an increase of the global activity of the animals. We will also present the analyses carried-out to monitor the immune response aga inst the transgcne. This poster establishes the fcasibility for whole body AAV-mediated o-sarcoglycan gene transfer as a therapeutic approach.

138. Ameliorating Dystrophic Pathology Via AAV-Mediated Gene Delivery of Myostatin Propeptide

Chunping Qlao ,' Jianbin Li,' Bing wang,' Juan Li,' Xiao Xiao.' 'Molecuar Pharmaceutics. UNC Shoo! ofPharmacy; Chapel Hill. NC; lDepartment a/Orthopaedic Surge/yo University a/Pittsburgh, Pittsburgh, PA.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is the most common, disabling and lethal muscle disease. Myostatin has been extensively documented as a negative regulator of muscle growth. Myostatin blockades therefore offers an effective strategy for treating a number of muscle degenerative diseases, including sarcopcnia and muscular dystrophy. In this study, we investigated whether gene delivery of myostatin inhibitors, specifically, the propeptide, could improve muscle growth and ameliorate the pathologies ofDMD in mouse model. The serotype 8 AAV-MPR076AFc vector was delivered into 3-month-old mdx mice by simple tail vein injection. The treated mdx mice started to gain weight two weeks after vector injection (p
139. Isolation, Characterization, and Myogenesis of Satellite Cells Derived from Skeletal Muscle Nicholas Icronimakis ,' Gayathri Balasundaram ,' Jeffrey S. Chamberlain.! Morayma Reyes. I /Pathology. University 0/ Washington. Seattle. IVA; lNeul'Ology. University ofWashington, Seattle. WA. Satellite cells constitute the natural stem cell reservoir for regeneration in adult skeletal muscle. The regenerative role and capability of satellite cells (SC) in skeletal muscle makes them a primed candidate for treatment of degenerative diseases such as muscular dystrophy. However, the challenge of isolation and expansion of pure satellite cell populations has encumbered their use in clinical applications. We report here the direct isolation of SC from skeletal muscles by fluorescence-activated cell sorting based on the expression of Sca-l , CD34 , CD31 and CD45 cell surface S54

antigens. Sca-I is predominantly a marker of hematopoietic stem cells and as we have reported a marker of endothelial cells in the skeletal muscle. Conveniently SC arc Sea-l negative. CD34 is also a marker of hematopoietic stem cells and some endothelial cells, but in the skeletal muscles CD34 is highly expressed by SC. By utilizing a host of antibodies we have isolated a very distinct and homogenous population recognized as Sea-L, CD31', CD34+ and CD45'from both wt and mdx (dystrophyn -/-) mice, ages ranging from newborn to 25 months. Because some hematopoietic and endothelial cells also express these markers we excluded all CD45 +cells (hematopoietic) and CD31+ (endothelial) cells. Furthermore, the forward and size scatter pattern ofthis population corresponds with the size morphology of SC as a hornogenously small mononuclear population. Depending on age, this population represents 0.5-2% of all mononuclear cells derived from skeletal muscles. The abundance ofSC in the skeletal muscle declines with age though more severely in mdx mice to almost undetectable levels by 25 month. RT-PCR analysis and immunohistochemistry offreshly sorted cells confirms this population expresses many satellite cell markers such as Pax7, NCAM, MyoD , Myf5 , Syndecan 3, CD34 , and c-rnet while lacking expression ofendothelial markersTEK, vWF, and FIt. Nearly 100% of these cells express Pax7 and Myf5 signifying this population to be very pure and homogenous. In addition, staining of freshly FACS-sorted SC for NCAM and c-met are polarized which correlates with polarization ofthese markers in muscle tissue sections towards the basal lamina. Interestingly, SC derived from mdx mice express lower levels ofNCAM, MyoD, Myf5 and syndecan 3, perhaps due to impaired myogenesis. We have culture-expanded these cells in vitro in FlOC with 15% horse serum and 10 nglml bFGF, obtaining clones of more than 1000 SC (>10 cell doublings) for seven days. These cells can differentiate into robust myotubes when bFGF is withdrawn and horse serum is reduced to 1.5%. To demonstrate the myogenic potential ofthcsc cells, Sea-l', CD3I', CD45', CD34+cells were FACS-sorted from GFP mice and directly injected intramuscularly in the tibialis anterior muscles ofmdx mice. Two weeks after transplantation multiple green myofibers were seen throughout the TA muscle , demonstrating the regenerative capability ofthese cells. This new approach using flow eytometry to directly isolate SC will be extremely useful in study ing their biology for the development of regenerative treatments for muscular diseases.

140. Gait in BioTO-2 and Bio14.6 Dystrophic Hamsters

Thomas G. Hampton.! Ivo Amende,' Ajit Kale, 2 Scott Mccue.l Hemmi N. Bhagavan,' Anton H. M. Terpstra,' Case Vanfrongen.' JR & D. Biobreeders Inc.• Watertown, MA; lR & D. Mousespecif-

ics Inc.. Boston, MA.

The delta-sarcoglycan-deflcient hamster strains BlO 14.6 and BlO T02 are excellent models to study muscular dystrophy and the efficacy ofgene therapy. Gait disturbances, important clinically, have not yet been described in these hamster models. Accordingly, we compared the gait of BIO 14,6 (n=12) and BIO T02 (n=12) dystrophic hamsters to healthy BIO FIB (n=12) control hamsters. We used ventral plane videography to determine gait indices in 3mo and 9-mo old male BlO 14.6, BIO T02, and BIO FIB hamsters walking on a transparent treadmill belt at 16 cm/s. Gait indices were based on -10 consecutive strides for each of the 4 limbs. We also studied l-mo old BlOT02 (n=4) and FIB (n=4) hamsters and found kinematic and postural changes in both BlO 14.6 and BlO T02 hamsters, including significantly shorter swing, stride, and stance durations. Stride length was -13% shorter (P
© The American $<)(;(1,: 1)" o f Gene Therapy

(303 ± II ms; 1'<0.05). Braking duration, reflecting generation of ground reaction forces , was delayed in 9-mo old BlO 14.6 (142 ± 7 ms) and BlOT02 hamsters(185 ±9 ms)compared to BlO FIB (112 ± 14 ms; 1'<0.05). 1·lind paw eversion, evidence of muscle weakness, was greater in 9-mo old BlO 1'02 than in BlO FI B hamsters (20.1 ± 1.10 vs, 6.7 ± 1.70 ; 1'<0.05). The propulsive deficit was apparent in BlO 14.6 and BlO 1'02 at 3-mo and in BlO 1'02 animals at I-mo of age. Our quantitative analysis of gait demonstrates gait disturbances in hamsters with muscular dystrophy that commence as early as I-mo of age. Our findings show that these dystrophic BlO hamsters recapitulate the functional features of human muscular dystrophy. Early detection and quantitative measures of these gait abnormalities will accelerate the development of gene therapy for treating muscular dystrophy.

141. GNE Vector Development and Testing for Gene Replacement Treatment of Hereditary Inclusion Body Myopathy Chris Jay,' ·2 Stephan Hinderlich,' Daniel Dervish,' Gregory Nemunaitis," John 1. Nernunaitis.P? Phillip B. Maples.':' 'Cancer Research. Murex Pharmaceuticals, lnc., Dallas, TX; lCancer Research, Mary Crowley Cancer Research Centers, Dallas, T}(,' JClwrite Universitatsmedizln, Benjamin Franklin Institut fiir Biochemie und Molekularbiologie, Berlin-Dahlem, Germany; "Research. ffIBM Research Group, Encino, CA; sMedical, Metrohealth Medical Center; Cleveland, Off; "Cancer Research, Baylor Sammons Cancer Center; Dallas, T}(,' "Cancer Research, Texas Oncology, 1'.;/., Dallas, TX. Hereditary inclusion body myopathy (HIBM) is a disease which causes severe skeletal muscle wasting, and leads to almost complete disability as early as ages 35 to 45. There are no proven treatments for HIBM. Development ofthis disease is related to familial passage of a mutation ofthe ONE gene, which encodes the bifunctional enzyme UDP-OIeNAc 2-Epimerase / ManNAc Kinase (ONE/MNK). This is the rate limiting bifunctional enzyme that catalyzes the first 2 steps of sialic acid biosynthesis. Decreased sialic acid production consequently leads to decreased sialyation of a variety of glycoproteins including critical muscle protein alpha-dystroglycan (a-DO). This in tum severely cripples muscle function and appears to lead to the onset of the syndrome. We hypothesize that replacing the mutated ONE gene with the normal gene may restore minimal sialic acid synthesis production enabling improvement in muscle func tion and/or delay in rate of muscle deterioration. We have constructed four ONE gene/CMV promoter plasmids (encoding the wildtype, a negative mutant and two sialuria forms ofGNE) and demonstrated enhanced ONE gene activity following delivery to GNE-minus Lec3 cells. This revealed that replacement with wild type ONE cDNA restores ONE/MNK enzyme function . We have assayed both the epimerase and kinase activities of ONE from transfected and control Lec3 cells and found that Lec3 cell transfected with wildtype ONE have a similar sialic acid content to untransfected Lec3 cells. Untransfected Lec3 cells bypass the block in sialic acid biosynthesis by utilizing components in fetal bovine serum. Interestingly, the two ONE sialuria mutants had significantly higher levels ofsialic acid. We are now manufacturing and testing the ONE expression vector in a targeted Molecular Therapy volume 15.Supp lem ent I. ~I; 'r 2007 Co pr righ t © The American Soc,.; ety o f G ene Therapy

immunoliposomal nanoparticle (ONE-IL). Shortly, we will initiate in vivo toxicology studies and file our IND. In our Phase I trials, we intend to administer this complex intramuscularly and subsequently, intravenously, to test the safety and efficacy (enhanced or stabilized muscle function) of ONE gene replacement in HIBM.

142. Spliceosome-Mediated RNA TransSplicing for Muscular Dystrophies

Chunping Qiao,' Jianbin Li ,' Bing Wang.! Juan Li,' Xiao Xiao.' [Department ofMolecular Pharmaceutics, UNC School ofPharmacy. Chapel Hill, NC; lDepartmellt ofOrthopaedic Surgery; University ofPittsburgh, Pittsburgh, PA. Spliceosome-mediated RNA trans-splicing (SMaRT) is a novel RNA-reprogramming strategy. It offers several potential advantages over traditional gene replacement therapy, such as reduced transgcne size and elimination of ectopic expression of the repaired product by retaining endogenous regulation. In this study, we explored the potential application of SMaRT technology for muscular dystrophies. We use the Duchenne muscular dystrophy mouse, the mdx , as a model system, in which the mutated gene dystrophin was shown to carl)' a premature stop codon in exon 23. We intended to usc pretrans-splicing molecules (PTM) containing mouse dystrophin intron 22 sequence linked the C-terminal coding region of dystrophin to intercept the RNA splicing before exon 23. The resultant is a transspliced mRNA encoding a functional dystrophin. To examine the trans-splicing efficiency in cultured cells, we first created a target plasmid by inserting the dystrophin intron 22 into mini-dystrophin gene 3858 , between Rod 2 (R2) and Rod 22 (R22) regions, generating plasmid pAAV-cmv-3858-spolyA-in22. We then constructed three PTMs containg different binding domains of intron 22 and the C-half of the mini-dystrophin 3990 with a myc tag. The PTM plasmids were respectively co-transfected with the target plasmid for trans-splicing into 293 cells. Trans-splicing events were monitored by both RT-PCR and western blot. Sequencing results of RT-PCR fragment confirmed the expected trans-splicing products, while western blot showed expected myc -tagged mini-dystrophin, We then packaged those three PTMs into adeno-associated viral vector and injected them into the leg muscle of three-month-old mdx mice. Two months after vector injection, the mice were sacrificed and the injected muscles were carefully dissected. The trans-splicing events were analyzed by both immunofluorescent (IF) staining and RT-PCR. IF staining indicated low percentage of mini-dystrophin positive cells, while RT-PCR failed to detect trans-splicing product. Our results indicated that SMaRT technology offers some potential for the treatment of muscular dystrophies; however, its efficiency has to be improved to render therapeutic effect.

143. Effect of Insulin-Like Growth Factor-1 (IGF-1) on Global Gene Expression Profiles in Murine Skeletal Muscle C. Ramana Bhasker, Shaochun Song, Theodore Friedmann. [Centerfor Molecular Genetics, Dept. ofPediatrics, University of California San Diego, La Jolla, CA. The peptide hormone IGFI is a powerful mitogen that plays vital roles in the action of growth hormone and is a central participant in growth and function of skeletal muscle. It has been shown to bring about important therapeutic effects in the mdx mouse model of muscular dystrophy and other forms of muscular and neuromuscular disease such as a mouse model of ALS. There is little detailed understanding of the global effects oflOF-1 on global patterns of gene expression. To develop a more thorough understanding of the we have recently undertaken mimechanisms of action ofIGF~I, croarray-based and protcomic studies oflOF-1 on murine myoblasts in culture and in skeletal muscle in vivo. We report here a descripS55