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942. Electrocardiographic and Pathologic Improvement of mdx Heart by Transduction with rAAV9-Microdystrophin
MUSCLE AND CONNECTIVE TISSUE - MUSCLE DISEASE DIRECT GENE THERAPY vesicles, important contributory organelles in membrane repair. The membrane patch is facilitated by binding and trafficking proteins that include annexins, calpain, and affixins. Two dysferlin deficient mouse models, SJL and A/J, provide an opportunity to test translational treatment approaches. Dysferlin is a large 237 kDa protein with a single transmembrane domain and seven C2 domains with unknown function. The size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8). Recent findings demonstrating that rAAV5 capsid packages up to 8.9 kb [Alloca, M et al. (2008)], prompted us to generate a vector containing full-length DYSF. Potential advantages of a full cDNA versus a truncated transgene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. Herein we describe our in vivo work comparing dysferlin gene expression in SJL and AJ mice. We generated a cassette containing the DYSF cDNA driven by the muscle specific MHCK7 promoter. A chimeric intron was added to augment RNA processing. The cassette was packaged into an AAV2/5 vector using standard triple transfection and purified using iodixanol gradients and ion exchange chromatography. We injected the tibialis anterior (TA) muscle of 4-6 week old SJL and A/J mice with rAAV5.MHCK7.DYSF at low (3 x 1010 vg) and high (1011 vg) doses. Animals were sacrificed after 4 weeks and muscle sections were immunostained for dysferlin expression. Transduction was achieved in a dose dependent manner in both AJ and SJL mice with more robust expression in AJ mice. Western blot analysis confirmed the immunostaining results. Treated AJ mice had a clear 237kDa band which was absent in control animals. Band intensity correlated with dose. SJL mice express ∼15% residual dysferlin protein, which increased by gene transfer. These results provide proof of principle that a full-length dysferlin cDNA can be delivered efficiently to muscle using the AAV5 serotype. Ongoing efficacy studies will help define required dosing levels for functional benefit translatable to LGMD2B and MM patients. M. Allocca et al. (2008). Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. J Clin Invest 118:1955-64.
942. Electrocardiographic and Pathologic Improvement of mdx Heart by Transduction with rAAV9-Microdystrophin
Jin-Hong Shin,1,2 Sachiko Ohshima-Hosoyama,1,3 Hiromi Kinoh,1 Takashi Okada,1 Shin’ichi Takeda.1 1 Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; 2Neurology, Graduate School of Medicine, Pusan National University, Busan, Republic of Korea; 3Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Background: Duchenne muscular dystrophy (DMD) is an inherited disorder by the defective dystrophin and characterized by progressive deterioration of skeletal and cardiac muscles. Cardiomyopathy is a significant burden in advanced DMD patients and a leading cause of deaths. Electrocardiographic (ECG) abnormalities appear in almost all DMD patients. Common ECG abnormalities in DMD patients include tachycardia, shortened PR interval, and reversed R/S ratio. We utilized mdx mice, which is the representative animal model of DMD, and also show ECG abnormalities corresponding to those of DMD patients. AAV9 vector was used for systemic delivery of the microdystrophin. We monitored the therapeutic effects focusing on ECG, and tried to clarify the mechanism. Methods: Four weeks old mdx mice were transduced with the rAAV9-microdystrophin driven by the CMV promoter (3.0 x 1012 v.g./body) via the tail vein. Four, 8, and 24 weeks after transduction, the ECG were taken under anesthesia, and compared with those of age-matched C57BL10 mice and untransduced mdx mice. Autonomic blockade was performed S360
with intraperitoneal injection of either atropine (0.5 mg/kg body weight) or propranolol (1.0 mg/kg). Mice were then sacrificed for histologic analysis. Results: Immunofluorescence staining revealed that more than 90% of the cardiac myofibers were transduced with the microdystrophin. As for the abnormalities in rhythm, heart rate (HR) was higher and PR interval was shorter (P<0.01) in the untransduced mdx mice than C57BL10 mice, while there was no significant difference between C57BL10 mice and transduced mdx mice. QRS interval lengthened and S/R ratio decreased significantly (P<0.01) in untransduced mdx mice compared to C57BL10 mice after 8 weeks old, but the transduced mdx mice did not show such abnormalities even at 24 weeks after transduction. Parasympathetic blockade by atropine caused increase in HR in the transduced mdx mice similar to that of C57BL10 mice, while untransduced mdx mice remained to show paradoxical HR drop. Quantification of cardiac fibrosis revealed that fibrous contents and level of TGF-β expression is significantly increased in untransduced mdx mice at their age of 28 weeks, but with the transduced mdx mice it was not significantly different from that of C57BL10 mice. Conclusion: Although cardiac pathology in untransduced mdx mice is not evident before 12 weeks of age, we could detect several abnormalities in the ECG profiles, as early as 8 weeks old. Our results indicate that ECG changes in rhythm reflect ongoing cardiac failure, mediated most likely by parasympathetic nervous system, while changes in QRS complex are correlated with the pattern of cardiac fibrosis. Improvement in both aspects of ECG abnormalities by microdystrophin transduction suggests its therapeutic effects against dystrophin-deficient cardiomyopathy.
943. Persistent Expression of FLAG Tagged Micro-Dystrophin in Non-Human Primates with Intramuscular and Vascular Delivery
Louise R. Rodino-Klapac,1 Chrystal L. Montgomery,1 William G. Bremer,1 Vinod Malik,1 Kim M. Shontz,1 Nancy Davis,1 Spencer Sprinkle,1 Katherine J. Campbell,1 Zarife Sahenk,1 K. Reed Clark,1 Christopher M. Walker,1 Louis G. Chicoine,1 Jerry R. Mendell.1 1 The Research Institute at Nationwide Children’s Hospital, Columbus, OH. Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Since a primate model for the disease is not available, it is difficult to design pre-clinical studies that accurately predict clinical outcomes. A critical issue in translation is vector delivery across the vascular barrier. While this has been achieved in the mdx mouse using AAV, successfully translating rodent studies to patients remains a challenge. Dosing issues are paramount in scaling gene delivery from small animals to human patients. In addition, from a safety perspective, regional vascular delivery (e.g., one limb) rather than perfusing the whole animal is more acceptable. Some have proposed intravenous delivery in the limb against a high pressure gradient to facilitate crossing the vascular barrier. In our ongoing studies we have focused on a more physiological gene delivery strategy through the arterial circulation with considerable success in mice and monkeys. Our prior primate studies used green fluorescent protein (GFP) to show the distribution of transduced muscle fibers. A more relevant marker for pre-clinical studies that focuses on DMD is a micro-dystrophin FLAG-tagged protein, whereby an 8 amino acid epitope tag is fused to the C-terminus. We first tested this construct by intramuscular injection of the tibialis anterior (TA) in three rhesus macaques with 5 x 1012 vg of AAV8.MCK.micro-dys.FLAG. Saline was injected into the contralateral TA. At 8 weeks post gene delivery all three non-human primates exhibited robust gene expression with 60-80% of TA muscle fibers expressing micro-dystrophin-FLAG. At 5 months persistent micro-dystrophin gene expression was seen in up to 75% of fibers. Western blot analysis confirmed the 138 kDa micro-dys. Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
942. Electrocardiographic and Pathologic Improvement of mdx Heart by Transduction with rAAV9-Microdystrophin
Jin-Hong Shin,1,2 Sachiko Ohshima-Hosoyama,1,3 Hiromi Kinoh,1 Takashi Okada,1 Shin’ichi Takeda.1 1 Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; 2Neurology, Graduate School of Medicine, Pusan National University, Busan, Republic of Korea; 3Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Background: Duchenne muscular dystrophy (DMD) is an inherited disorder by the defective dystrophin and characterized by progressive deterioration of skeletal and cardiac muscles. Cardiomyopathy is a significant burden in advanced DMD patients and a leading cause of deaths. Electrocardiographic (ECG) abnormalities appear in almost all DMD patients. Common ECG abnormalities in DMD patients include tachycardia, shortened PR interval, and reversed R/S ratio. We utilized mdx mice, which is the representative animal model of DMD, and also show ECG abnormalities corresponding to those of DMD patients. AAV9 vector was used for systemic delivery of the microdystrophin. We monitored the therapeutic effects focusing on ECG, and tried to clarify the mechanism. Methods: Four weeks old mdx mice were transduced with the rAAV9-microdystrophin driven by the CMV promoter (3.0 x 1012 v.g./body) via the tail vein. Four, 8, and 24 weeks after transduction, the ECG were taken under anesthesia, and compared with those of age-matched C57BL10 mice and untransduced mdx mice. Autonomic blockade was performed S360
with intraperitoneal injection of either atropine (0.5 mg/kg body weight) or propranolol (1.0 mg/kg). Mice were then sacrificed for histologic analysis. Results: Immunofluorescence staining revealed that more than 90% of the cardiac myofibers were transduced with the microdystrophin. As for the abnormalities in rhythm, heart rate (HR) was higher and PR interval was shorter (P<0.01) in the untransduced mdx mice than C57BL10 mice, while there was no significant difference between C57BL10 mice and transduced mdx mice. QRS interval lengthened and S/R ratio decreased significantly (P<0.01) in untransduced mdx mice compared to C57BL10 mice after 8 weeks old, but the transduced mdx mice did not show such abnormalities even at 24 weeks after transduction. Parasympathetic blockade by atropine caused increase in HR in the transduced mdx mice similar to that of C57BL10 mice, while untransduced mdx mice remained to show paradoxical HR drop. Quantification of cardiac fibrosis revealed that fibrous contents and level of TGF-β expression is significantly increased in untransduced mdx mice at their age of 28 weeks, but with the transduced mdx mice it was not significantly different from that of C57BL10 mice. Conclusion: Although cardiac pathology in untransduced mdx mice is not evident before 12 weeks of age, we could detect several abnormalities in the ECG profiles, as early as 8 weeks old. Our results indicate that ECG changes in rhythm reflect ongoing cardiac failure, mediated most likely by parasympathetic nervous system, while changes in QRS complex are correlated with the pattern of cardiac fibrosis. Improvement in both aspects of ECG abnormalities by microdystrophin transduction suggests its therapeutic effects against dystrophin-deficient cardiomyopathy.
943. Persistent Expression of FLAG Tagged Micro-Dystrophin in Non-Human Primates with Intramuscular and Vascular Delivery
Louise R. Rodino-Klapac,1 Chrystal L. Montgomery,1 William G. Bremer,1 Vinod Malik,1 Kim M. Shontz,1 Nancy Davis,1 Spencer Sprinkle,1 Katherine J. Campbell,1 Zarife Sahenk,1 K. Reed Clark,1 Christopher M. Walker,1 Louis G. Chicoine,1 Jerry R. Mendell.1 1 The Research Institute at Nationwide Children’s Hospital, Columbus, OH. Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Since a primate model for the disease is not available, it is difficult to design pre-clinical studies that accurately predict clinical outcomes. A critical issue in translation is vector delivery across the vascular barrier. While this has been achieved in the mdx mouse using AAV, successfully translating rodent studies to patients remains a challenge. Dosing issues are paramount in scaling gene delivery from small animals to human patients. In addition, from a safety perspective, regional vascular delivery (e.g., one limb) rather than perfusing the whole animal is more acceptable. Some have proposed intravenous delivery in the limb against a high pressure gradient to facilitate crossing the vascular barrier. In our ongoing studies we have focused on a more physiological gene delivery strategy through the arterial circulation with considerable success in mice and monkeys. Our prior primate studies used green fluorescent protein (GFP) to show the distribution of transduced muscle fibers. A more relevant marker for pre-clinical studies that focuses on DMD is a micro-dystrophin FLAG-tagged protein, whereby an 8 amino acid epitope tag is fused to the C-terminus. We first tested this construct by intramuscular injection of the tibialis anterior (TA) in three rhesus macaques with 5 x 1012 vg of AAV8.MCK.micro-dys.FLAG. Saline was injected into the contralateral TA. At 8 weeks post gene delivery all three non-human primates exhibited robust gene expression with 60-80% of TA muscle fibers expressing micro-dystrophin-FLAG. At 5 months persistent micro-dystrophin gene expression was seen in up to 75% of fibers. Western blot analysis confirmed the 138 kDa micro-dys. Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy