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139. Isolation, Characterization, and Myogenesis of Satellite Cells Derived from Skeletal Muscle
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
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