- Email: [email protected]
137. Global Force Improvement by Systemic AAV Delivery of α-Sarcoglycan Transgene in Deficient Mice
also tested how cxDB3 might interact with the DOC in vitro and in vivo utilizing coimmunoprecipitation assays . Muscles throu ghout cxDB-I- mice were transduced with rAAV6-DB2 and rAAV6-DB3. cxDB2 and cxDB3 both localized to the sarcolemma and were concentrated within the folds of the myotendinous and neuromuscular junctions. cxDb3 and cxDb-2 were equally effective at preventing central nucleation in most skeletal muscle fibers, preventing fragmentation of neuromuscular synapses and restoring folds at the myotendinous junction. cxDB2 was more effective than cxDB3 in preventing fingerlike projections from protruding out ofthe synaptic borders. In coimmunoprecipitation studies, cxDb-3 was demonstrated to interact with the sareoglycan subcomplex through an individual sarcoglycan interaction in vitro. In vivo coimmunoprecipitation ofcxDB3 from whole muscle Iysates resulted in the majority of the DOC components being detected. cxDb-3 is a significant component of the DOC that functions to prevent muscle degene ration, synapse destabilization and myotenindous junction defects within skeletal muscle. These results suggest the reduction of cxDB3 from the sarcolemma in various muscular dystrophies likely contributes to their pathogenesis in a similar manner to cxDB I and cxDB2.
135. rAAV Type 8-Mediated Extensive Therapeutic Gene Delivery into Skeltal Muscle of a-Sarcoglycan Deficient Mice Akiyo Nishiyama,' Beryl N. Arnpong,' Jin-Hong Shin ,' Hiroyuki Nakai,' Takashi Okada, I Shin'ichi Takeda.' 'Molecular Therapy; National Institute ofNeuroscience, NCN?, Kodaira, Tokyo, Japan; 'Molecular Genetics and Biochemistry, University 0/ Pittsburgh School ofMedicine, Pittsburgh, PA. Backgrounds: Autosomal recessive limb-girdle muscular dystrophy type 2D (LOMD2D) is caused by the mutations in the ti-sarcoglycan gene (cx-SG) and the absence of cx-SO results in a reduction ofwhole SO complex, composed of'u-, ~-, y-, o-SO, from the sarcolemma. The recombinant adeno-associated virus (rAAV) is expected to be useful to transduce cx-SO cDNA. However, the transduction by the rAAV type 2 (rAAV2) encoding o-sc cDNA showed cytotoxicity with immune response in the cx-SG-deficient mice. The rAAV2 only allows transduction of the gene through local injection, but the rAAV type 8 (rAAV8) enables systemic deliverly of the gene through intravenous injection. We examined the therapeutic effects of the rAAV8 encoding the cx-SO eDNA on the cx-SG-deficient mice. Methods: We generated the rAAV2 or rAAV8 encoding the cx-SO cDNA driven by a CMV promoter (rAAV2-cx-SO or rAAV8-cx-SO). We injected the rAAVs into the anterior tibial (TA) muscle of the cx-SG-defieient mice. The neonatal or adult cx-SG-deficient mice were injected with the ) x )0" vg or 5 x 10" vg of rAAVs, respectively. Moreover, we injected the rAAV8-cx-SO (5 x 1012 vg) into the tail vein of the 5-week-old cSG-deficient mice for systemic delivery. Results and Discussion: At 4 weeks after the injection of the rAAV8-cx-SG into cx-SG-deficient TA muscle, expression of cx-SO was widely distributed in the posterior leg muscles, including the TA, extensor digitorum longus , gastrocnemius, soleus, and tibialis posterior muscles . In particular, the rAAV8-cx-SG effectively transduced the posterior leg muscles as well as the heart of the adult mice. In contrast, the rAAV2-cxSO merely achieved local transduction of muscle fibers in the TA muscle. Together with wide expression ofcx-SO,expression ofother SOs was also detected in the posterior leg after the local injection ofthc rAAV8-cx-SO.The rAAV8-cx-SO-transduecd muscle did not induce the severe immune response or cytotoxicity after the low dose injection ofthe rAAV8-cx-SG. In association with the rAAV8-cx-SG transduction, the cx-SG-deficient muscle significantly decreased the degeneration-regeneration cycle. Furthermore, the contractile force of the rAAV8-CX-SO-injected muscle has been largely improved compared to the non-injected muscles. By systemic delivery of the Molecular Therapy Volume 15. Supp lement 1,.\ Iay2007 Copyright
~
Th e American Society or Gen e Th erapy
rAAV8-cx-SO into the adult cx-SG-deficient mice, we observed the cx-SO expression in the various tissues including the skeletal muscle, cardiac muscle , and liver at 4 weeks. Conclusion : The rAAV8 is an effective tool to deliver therapeutic genes into the dystrophic skeletal muscle and enables wide distribution of the gene . This extensive rAAV8-mediated cx-SG transduction in the limb-girdle muscular dystrophy type 2D model mice would pave the way for the future clinical application.
136. Viral Based Therapeutic for LGMD Type 20 Deficiencies Demonstrates Long-Term Results in Alpha-Sarcoglycan Deficient Animals Louise R. Rodino-Klapac,' Christopher J. Shilling,' Zarife Sahenk,' K. Reed Clark,' Jerry R. Mendell.' 'Center for Gene Therapy, Columbus Children s Research Institute, Columbus, 011. Limb-girdle muscular dystrophy (LOMD) type 2D is characterized by skeletal muscle weakness and results from mutations occurring in the cc-sarcoglycan gene. Localized in the sarcolemma, the sarcoglycans (cx ,~,y , and (5) arc a subcomplex ofthe dystrophinassociated proteins (DAP). Alpha-sarcoglycan (cx-SG)deficiency is the most common form of sarcoglycan-LGMD and no therapeutic treatments are currently available. The cx-SO mouse model provides an opportunity to test translational treatment approaches. Prior studies have suggested that AAV-mediated cx-SO gene transfer could not sustain expression because of transgene toxicity, potentially precluding clinical gene transfer tor LOMD2D. Herein , we describe our in vivo work comparing a -SG gene expression from either the ubiquitously expressed cytomegalovirus (CMV) promoter, or muscle specific promoters, muscle creatine kinase (MCK) and desmin (DES) in the cx-SO KO mouse in the context ofrAAV gene delivery. We injected the tibialis anterior (TA) muscle of4-6 week old cx-SOKO mice with rAAV\.cx-SO (CMV, MCK, DES promoter) at low (3 x 109vg) and high (3 x IOlOvg) doses. Sustained gene expression was observed irrespective of promoters at six and twelve weeks post gene transfer. Quantitation ofa-SG gene expression by fiber counts yielded similar levels ofmyofiber transduction for CMV and MCK at the 6 week high dose, 6).4% versus 64.4% respectively. However, cx-SO expression using the DES promoter was significantly lower at the high dose with 34% ofthe myofibers transduced. Similar levels of expression were seen at the 12 week time point for MCK and DES , while CMV exhibited a 25% reduction in expression. Our studies show sustained and robust gene expression well beyond the time at which transgene cytotoxic effects were previously reported using rAAV2. CMY.a-SG. Mononuclear cell analysis in our studies showed no evidence of infiltrating B or T cell subsets or macro phages . Differences in our findings compared to previous work could relate to AAV serotype. In summary, our data demonstrate robust a-SO gene expression as long as 3 months using AAV) , with no reduction in expression using the muscle specific MCK promoter. These findings enhance the possibility for gene therapy as a potential treatment option for LOMD2D and support our current efforts for launching a gene therapy study tor this disease.
137. Global Force Improvement by Systemic AAV Delivery of a-Sarcoglycan Transgene in Deficient Mice Marc Bartoli ,' Francoise Fougerousse,' Jerome Poupiot,' Olivier Danos,' Isabelle Richard.' /LGMD, Genethon, Evry, France. a-sarcogIycanopathy (Limb Girdle Muscular Dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by dcfieiency in a-sarcoglycan, a transmembrane protein part of the dystrophinassociated complex. We constructed recombinant adeno-associated S53
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
135. rAAV Type 8-Mediated Extensive Therapeutic Gene Delivery into Skeltal Muscle of a-Sarcoglycan Deficient Mice Akiyo Nishiyama,' Beryl N. Arnpong,' Jin-Hong Shin ,' Hiroyuki Nakai,' Takashi Okada, I Shin'ichi Takeda.' 'Molecular Therapy; National Institute ofNeuroscience, NCN?, Kodaira, Tokyo, Japan; 'Molecular Genetics and Biochemistry, University 0/ Pittsburgh School ofMedicine, Pittsburgh, PA. Backgrounds: Autosomal recessive limb-girdle muscular dystrophy type 2D (LOMD2D) is caused by the mutations in the ti-sarcoglycan gene (cx-SG) and the absence of cx-SO results in a reduction ofwhole SO complex, composed of'u-, ~-, y-, o-SO, from the sarcolemma. The recombinant adeno-associated virus (rAAV) is expected to be useful to transduce cx-SO cDNA. However, the transduction by the rAAV type 2 (rAAV2) encoding o-sc cDNA showed cytotoxicity with immune response in the cx-SG-deficient mice. The rAAV2 only allows transduction of the gene through local injection, but the rAAV type 8 (rAAV8) enables systemic deliverly of the gene through intravenous injection. We examined the therapeutic effects of the rAAV8 encoding the cx-SO eDNA on the cx-SG-deficient mice. Methods: We generated the rAAV2 or rAAV8 encoding the cx-SO cDNA driven by a CMV promoter (rAAV2-cx-SO or rAAV8-cx-SO). We injected the rAAVs into the anterior tibial (TA) muscle of the cx-SG-defieient mice. The neonatal or adult cx-SG-deficient mice were injected with the ) x )0" vg or 5 x 10" vg of rAAVs, respectively. Moreover, we injected the rAAV8-cx-SO (5 x 1012 vg) into the tail vein of the 5-week-old cSG-deficient mice for systemic delivery. Results and Discussion: At 4 weeks after the injection of the rAAV8-cx-SG into cx-SG-deficient TA muscle, expression of cx-SO was widely distributed in the posterior leg muscles, including the TA, extensor digitorum longus , gastrocnemius, soleus, and tibialis posterior muscles . In particular, the rAAV8-cx-SG effectively transduced the posterior leg muscles as well as the heart of the adult mice. In contrast, the rAAV2-cxSO merely achieved local transduction of muscle fibers in the TA muscle. Together with wide expression ofcx-SO,expression ofother SOs was also detected in the posterior leg after the local injection ofthc rAAV8-cx-SO.The rAAV8-cx-SO-transduecd muscle did not induce the severe immune response or cytotoxicity after the low dose injection ofthe rAAV8-cx-SG. In association with the rAAV8-cx-SG transduction, the cx-SG-deficient muscle significantly decreased the degeneration-regeneration cycle. Furthermore, the contractile force of the rAAV8-CX-SO-injected muscle has been largely improved compared to the non-injected muscles. By systemic delivery of the Molecular Therapy Volume 15. Supp lement 1,.\ Iay2007 Copyright
~
Th e American Society or Gen e Th erapy
rAAV8-cx-SO into the adult cx-SG-deficient mice, we observed the cx-SO expression in the various tissues including the skeletal muscle, cardiac muscle , and liver at 4 weeks. Conclusion : The rAAV8 is an effective tool to deliver therapeutic genes into the dystrophic skeletal muscle and enables wide distribution of the gene . This extensive rAAV8-mediated cx-SG transduction in the limb-girdle muscular dystrophy type 2D model mice would pave the way for the future clinical application.
136. Viral Based Therapeutic for LGMD Type 20 Deficiencies Demonstrates Long-Term Results in Alpha-Sarcoglycan Deficient Animals Louise R. Rodino-Klapac,' Christopher J. Shilling,' Zarife Sahenk,' K. Reed Clark,' Jerry R. Mendell.' 'Center for Gene Therapy, Columbus Children s Research Institute, Columbus, 011. Limb-girdle muscular dystrophy (LOMD) type 2D is characterized by skeletal muscle weakness and results from mutations occurring in the cc-sarcoglycan gene. Localized in the sarcolemma, the sarcoglycans (cx ,~,y , and (5) arc a subcomplex ofthe dystrophinassociated proteins (DAP). Alpha-sarcoglycan (cx-SG)deficiency is the most common form of sarcoglycan-LGMD and no therapeutic treatments are currently available. The cx-SO mouse model provides an opportunity to test translational treatment approaches. Prior studies have suggested that AAV-mediated cx-SO gene transfer could not sustain expression because of transgene toxicity, potentially precluding clinical gene transfer tor LOMD2D. Herein , we describe our in vivo work comparing a -SG gene expression from either the ubiquitously expressed cytomegalovirus (CMV) promoter, or muscle specific promoters, muscle creatine kinase (MCK) and desmin (DES) in the cx-SO KO mouse in the context ofrAAV gene delivery. We injected the tibialis anterior (TA) muscle of4-6 week old cx-SOKO mice with rAAV\.cx-SO (CMV, MCK, DES promoter) at low (3 x 109vg) and high (3 x IOlOvg) doses. Sustained gene expression was observed irrespective of promoters at six and twelve weeks post gene transfer. Quantitation ofa-SG gene expression by fiber counts yielded similar levels ofmyofiber transduction for CMV and MCK at the 6 week high dose, 6).4% versus 64.4% respectively. However, cx-SO expression using the DES promoter was significantly lower at the high dose with 34% ofthe myofibers transduced. Similar levels of expression were seen at the 12 week time point for MCK and DES , while CMV exhibited a 25% reduction in expression. Our studies show sustained and robust gene expression well beyond the time at which transgene cytotoxic effects were previously reported using rAAV2. CMY.a-SG. Mononuclear cell analysis in our studies showed no evidence of infiltrating B or T cell subsets or macro phages . Differences in our findings compared to previous work could relate to AAV serotype. In summary, our data demonstrate robust a-SO gene expression as long as 3 months using AAV) , with no reduction in expression using the muscle specific MCK promoter. These findings enhance the possibility for gene therapy as a potential treatment option for LOMD2D and support our current efforts for launching a gene therapy study tor this disease.
137. Global Force Improvement by Systemic AAV Delivery of a-Sarcoglycan Transgene in Deficient Mice Marc Bartoli ,' Francoise Fougerousse,' Jerome Poupiot,' Olivier Danos,' Isabelle Richard.' /LGMD, Genethon, Evry, France. a-sarcogIycanopathy (Limb Girdle Muscular Dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by dcfieiency in a-sarcoglycan, a transmembrane protein part of the dystrophinassociated complex. We constructed recombinant adeno-associated S53
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