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583. Baculovirus-Engineered Mesenchymal Stem Cells Heal the Critical-Sized Femoral Segmental Bone Defects in Rabbits
MUSCULO-SKELETAL GENE & CELL THERAPY 583. Baculovirus-Engineered Mesenchymal Stem Cells Heal the Critical-Sized Femoral Segmental Bone Defects in Rabbits
Chin-Yu Lin,1 Yu-Han Chang,2 Kun-Ju Lin,3 Tzu-Chen Yen,3 WenYi Lo,1 Chia-Hsin Luo,1 Ching-Lung Tai,4 Yu-Chen Hu.1 1 Department of Chemical Engineering, National Tsing Hua University, Hsin-Chu, Taiwan; 2Department of Orthopaedic, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 3Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 4Graduate Institute of Medical Mechatronics, Chang Gung University, Taoyuan, Taiwan. Management of massive segmental bone defects remains a challenging clinical problem and bone marrow-derived mesenchymal stem cells (BMSCs) hold promise for bone regeneration. To explore whether BMSCs engineered by baculovirus (an emerging gene delivery vector) can heal large bone defects, the New Zealand White (NZW) rabbit BMSCs were transduced with the BMP2-expressing baculovirus or VEGF-expressing baculovirus, co-seeded to scaffolds and co-implanted into critical-sized (10 mm) femoral segmental defects in NZW rabbits. The transduced cells expressed functional BMP2 and VEGF as conrmed by ELISA and functional assays. X-ray analysis revealed that the baculovirus-engineered BMSCs not only bridged the defects at as early as week 2, but also healed the defects in 100% of rabbits (13/13) at week 4. The osteogenic metabolism, as monitored by positron emission tomography (PET), rose initially but subsided at week 8, suggesting the completion of bone healing. When compared with other control groups, the BMP2/ VEGF-expressing BMSCs remarkably enhanced the segmental bone repair and mechanical properties, as evidenced by micro-computed tomography (µCT), histochemical staining and biomechanical testing. The immunohistochemical staining further attested that the ameliorated bone healing concurred with the augmented angiogenesis. These data altogether demonstrated, for the rst time, that BMSCs engineered to express BMP2 and VEGF synergistically accelerate the repair of large femoral bone defects and improve the quality of the regenerated bone, which paves a new avenue to utilizing baculovirus as a vector for BMSCs modication and regenerative medicine.
584. Genetic Evidence for a Structural Role of Į–dystrobrevin-3 within the DystrophinGlycoprotein Complex
Guy L. Odom,1 Glen B. Banks,1 Dewayne Townsend,2 Marvin E. Adams,3 Joseph M. Metzger,2 Stanely C. Froehner,3 Jeffrey S. Chamberlain.1 1 Neurology, University of Washington, Seattle, WA; 2Integrative Biology & Physiology, University of Minnesota, Minneapolis, MN; 3 Physiology and Biophysics, University of Washington, Seattle, WA. α-Dystrobrevin (αDB) is an integral component of the dystrophinglycoprotein complex (DGC) at the sarcolemma of myobers. Mice lacking αDB1-3 (adbn-/-) in striated muscle present with several phenotypic changes consistent with dystrophy including myopathy, prominent neuromuscular and myotendinous junction defects, and a mild cardiomyopathy. Decreasing amounts of αDB from the sarcolemma contributes to the severity of disease in several muscular dystrophies including Duchenne muscular dystrophy (DMD). Both αDB1 and αDB2 transgenes restore the skeletal muscle abnormalities in the adbn-/- to varying degrees because of subtle differences in their location within the sarcolemma and varying functional (phosphorylation/protein-interaction) domains contained in each protein(Grady et al., 2003; Grady et al., 1999; Grady et al., 2000). Because αDB3 is the shorter isoform which lacks these functional domains, including being unable to bind directly to dystrophin, it has previously been assumed to play only a negligible role in the S226
DGC. Here, overall cardiac functional workload was assessed in wild type and mutant mice using a dobutamine stress test protocol followed by measurement of hemodynamic properties. We measured 18 ECG parameters at rest, and at 5 time points. None of these parameters showed signicant differences, suggesting a relatively normal electrical activity. We next analyzed the role of αDB3 in skeletal muscle by systemically delivering αDB3 to adbn-/- mice with recombinant adeno-associated virus serotype 6. The results demonstrated αDB3 to be an important DGC component within skeletal muscle, showing localization to the sarcolemma despite being unable to bind dystrophin, and a robust interaction with ankyrin as demonstrated by in vitro co-immunoprecipitation, suggesting a cooperative role toward the stabilization of myobers. Overall, expression in vivo of αDB3 resulted in the mitigation of prominent dystrophic abnormalities including the prevention of muscle degeneration, synapse fragmentation, and shallow folds within the myotendinous junction. These results suggest an important structural role of αDB3 whose absence can contribute to sarcolemma fragility in DMD and other muscular dystrophies.
585. rAAV8-Mediated Protein-Anchoring Therapy for Targeting Collagen Q-Tailed Acetylcholinesterase to the Neuromuscular Junction
Mikako Ito,1 Yumi Suzuki,1 Takashi Okada,2 Takayasu Fukudome,3 Toshiro Yoshimura,4 Akio Masuda,1 Shin’ichi Takeda,2 Eric Krejci,5 Kinji Ohno.1 1 Ctr for Neurological Diseases and Cancer, Nagoya Univ. Grad. Sch. of Med, Nagoya, Aichi, Japan; 2Dept of Molecular Therapy, NCNP, Kodaira, Tokyo, Japan; 3Dept of Neurology, Nagasaki Kawatana Med Ctr, Nagasaki, Nagasaki, Japan; 4Dept of Occupational Therapy, Nagasaki Univ. Grad. Sch. of Biomed Sci, Nagasaki, Nagasaki, Japan; 5U686, INSERM, Paris, France. Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defect of ColQ causes endplate AChE deciency. ColQ has a proprietary targeting signal for the synaptic basal lamina, and we can specically anchor ColQ-tailed AChE to a section of the NMJ in vitro. This observation prompted us to hypothesize that ColQ-tailed AChE expressed in a limited number of skeletal muscles can be propagated and anchored to the NMJs in Colq-/- mice. We thus constructed a recombinant adeno-associated virus serotype 8 (AAV8) carrying human COLQ. Intravenous administration of AAV8-COLQ normalized Colq-/- mice behaviorally, histochemically, morphologically, biochemically, and electrophysiologically. We also injected AAV8-COLQ intramuscularly to a single hindlimb, and found that ColQ-tailed AChE was expressed to ∼15% of the wild-type littermates even in non-injected limbs. We demonstrate that the tissuetargeting signal of an extracellular matrix molecule can be exploited to specically deliver the transgene product to the target tissue. A similar strategy can be potentially applied to a broad spectrum of congenital defects of extracellular matrix molecules.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Chin-Yu Lin,1 Yu-Han Chang,2 Kun-Ju Lin,3 Tzu-Chen Yen,3 WenYi Lo,1 Chia-Hsin Luo,1 Ching-Lung Tai,4 Yu-Chen Hu.1 1 Department of Chemical Engineering, National Tsing Hua University, Hsin-Chu, Taiwan; 2Department of Orthopaedic, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 3Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 4Graduate Institute of Medical Mechatronics, Chang Gung University, Taoyuan, Taiwan. Management of massive segmental bone defects remains a challenging clinical problem and bone marrow-derived mesenchymal stem cells (BMSCs) hold promise for bone regeneration. To explore whether BMSCs engineered by baculovirus (an emerging gene delivery vector) can heal large bone defects, the New Zealand White (NZW) rabbit BMSCs were transduced with the BMP2-expressing baculovirus or VEGF-expressing baculovirus, co-seeded to scaffolds and co-implanted into critical-sized (10 mm) femoral segmental defects in NZW rabbits. The transduced cells expressed functional BMP2 and VEGF as conrmed by ELISA and functional assays. X-ray analysis revealed that the baculovirus-engineered BMSCs not only bridged the defects at as early as week 2, but also healed the defects in 100% of rabbits (13/13) at week 4. The osteogenic metabolism, as monitored by positron emission tomography (PET), rose initially but subsided at week 8, suggesting the completion of bone healing. When compared with other control groups, the BMP2/ VEGF-expressing BMSCs remarkably enhanced the segmental bone repair and mechanical properties, as evidenced by micro-computed tomography (µCT), histochemical staining and biomechanical testing. The immunohistochemical staining further attested that the ameliorated bone healing concurred with the augmented angiogenesis. These data altogether demonstrated, for the rst time, that BMSCs engineered to express BMP2 and VEGF synergistically accelerate the repair of large femoral bone defects and improve the quality of the regenerated bone, which paves a new avenue to utilizing baculovirus as a vector for BMSCs modication and regenerative medicine.
584. Genetic Evidence for a Structural Role of Į–dystrobrevin-3 within the DystrophinGlycoprotein Complex
Guy L. Odom,1 Glen B. Banks,1 Dewayne Townsend,2 Marvin E. Adams,3 Joseph M. Metzger,2 Stanely C. Froehner,3 Jeffrey S. Chamberlain.1 1 Neurology, University of Washington, Seattle, WA; 2Integrative Biology & Physiology, University of Minnesota, Minneapolis, MN; 3 Physiology and Biophysics, University of Washington, Seattle, WA. α-Dystrobrevin (αDB) is an integral component of the dystrophinglycoprotein complex (DGC) at the sarcolemma of myobers. Mice lacking αDB1-3 (adbn-/-) in striated muscle present with several phenotypic changes consistent with dystrophy including myopathy, prominent neuromuscular and myotendinous junction defects, and a mild cardiomyopathy. Decreasing amounts of αDB from the sarcolemma contributes to the severity of disease in several muscular dystrophies including Duchenne muscular dystrophy (DMD). Both αDB1 and αDB2 transgenes restore the skeletal muscle abnormalities in the adbn-/- to varying degrees because of subtle differences in their location within the sarcolemma and varying functional (phosphorylation/protein-interaction) domains contained in each protein(Grady et al., 2003; Grady et al., 1999; Grady et al., 2000). Because αDB3 is the shorter isoform which lacks these functional domains, including being unable to bind directly to dystrophin, it has previously been assumed to play only a negligible role in the S226
DGC. Here, overall cardiac functional workload was assessed in wild type and mutant mice using a dobutamine stress test protocol followed by measurement of hemodynamic properties. We measured 18 ECG parameters at rest, and at 5 time points. None of these parameters showed signicant differences, suggesting a relatively normal electrical activity. We next analyzed the role of αDB3 in skeletal muscle by systemically delivering αDB3 to adbn-/- mice with recombinant adeno-associated virus serotype 6. The results demonstrated αDB3 to be an important DGC component within skeletal muscle, showing localization to the sarcolemma despite being unable to bind dystrophin, and a robust interaction with ankyrin as demonstrated by in vitro co-immunoprecipitation, suggesting a cooperative role toward the stabilization of myobers. Overall, expression in vivo of αDB3 resulted in the mitigation of prominent dystrophic abnormalities including the prevention of muscle degeneration, synapse fragmentation, and shallow folds within the myotendinous junction. These results suggest an important structural role of αDB3 whose absence can contribute to sarcolemma fragility in DMD and other muscular dystrophies.
585. rAAV8-Mediated Protein-Anchoring Therapy for Targeting Collagen Q-Tailed Acetylcholinesterase to the Neuromuscular Junction
Mikako Ito,1 Yumi Suzuki,1 Takashi Okada,2 Takayasu Fukudome,3 Toshiro Yoshimura,4 Akio Masuda,1 Shin’ichi Takeda,2 Eric Krejci,5 Kinji Ohno.1 1 Ctr for Neurological Diseases and Cancer, Nagoya Univ. Grad. Sch. of Med, Nagoya, Aichi, Japan; 2Dept of Molecular Therapy, NCNP, Kodaira, Tokyo, Japan; 3Dept of Neurology, Nagasaki Kawatana Med Ctr, Nagasaki, Nagasaki, Japan; 4Dept of Occupational Therapy, Nagasaki Univ. Grad. Sch. of Biomed Sci, Nagasaki, Nagasaki, Japan; 5U686, INSERM, Paris, France. Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defect of ColQ causes endplate AChE deciency. ColQ has a proprietary targeting signal for the synaptic basal lamina, and we can specically anchor ColQ-tailed AChE to a section of the NMJ in vitro. This observation prompted us to hypothesize that ColQ-tailed AChE expressed in a limited number of skeletal muscles can be propagated and anchored to the NMJs in Colq-/- mice. We thus constructed a recombinant adeno-associated virus serotype 8 (AAV8) carrying human COLQ. Intravenous administration of AAV8-COLQ normalized Colq-/- mice behaviorally, histochemically, morphologically, biochemically, and electrophysiologically. We also injected AAV8-COLQ intramuscularly to a single hindlimb, and found that ColQ-tailed AChE was expressed to ∼15% of the wild-type littermates even in non-injected limbs. We demonstrate that the tissuetargeting signal of an extracellular matrix molecule can be exploited to specically deliver the transgene product to the target tissue. A similar strategy can be potentially applied to a broad spectrum of congenital defects of extracellular matrix molecules.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy