- Email: [email protected]
174. A Novel Rationally Designed AAV Micro-Utrophin Vector Recruits nNOS to the Sarcolemma
MUSCULO-SKELETAL GENE & CELL THERAPY I 174. A Novel Rationally Designed AAV MicroUtrophin Vector Recruits nNOS to the Sarcolemma
Yi Lai,1 Junling Zhao,1 Yongping Yue,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO.
1
Sarcolemmal neuronal nitric oxide synthase (nNOS) is crucial for normal muscle function. Absence of sarcolemmal nNOS plays a critical role in the pathogenesis of muscular dystrophy. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Dystrophin anchors nNOS to the sarcolemma. Utrophin is a homolog of dystrophin and has been extensively investigated as a candidate gene for DMD gene therapy. Unfortunately, unlike dystrophin, utrophin cannot recruit nNOS to the sarcolemma. Inability of utrophin to restore sarcolemmal nNOS may significantly attenuate its therapeutic effect. To overcome this drawback of utrophin, we set to engineer novel utrophin genes that can fix nNOS to the muscle cell membrane. We recently found that dystrophin spectrin-like repeats 16 and 17 (R16/17) mediate sarcolemmal nNOS localization. Utrophin R15/16 shares great homology with utrophin R16/17. In this study, we examined whether replacing utrophin R15, or R16 or both with respective dystrophin R16 and R17 can result in novel utrophin genes capable of nNOS anchoring. A series of four repeats micro-utrophin AAV vectors were generated. All micro-utrophin genes contained a flag tag, utrophin N-terminus, utrophin repeats 1, and 22 and utrophin cysteine-rich domain. The two repeats between utrophin R1 and R22 were either original utrophin R15/16 or engineered with dystrophin R16/17 substitution. The micro-utrophin gene was packaged into AAV-9 and delivered AAV viruses to the tibialis anterior muscle of utrophin and dystrophin double knockout mice. One month after injection, we examined microgene expression and sarcolemmal nNOS localization. Robust expression was observed in all AAV infected muscle. As expected, the microgene construct carrying original utrophin R15/16 did not recover sarcolemmal nNOS. Constructs with either dystrophin R16 or R17 failed to restore nNOS to the sarcolemma. However when both repeats were replaced, sarcolemmal nNOS expression was restored. Our results suggest that both dystrophin R16 and R17 are required for nNOS localization. Further, dystrophin R16/17 is sufficient for sarcolemmal nNOS expression in the context of utrophin. The novel micro-utrophin gene described here may hold great promise for DMD gene therapy.
175. Determining the Therapeutic Window: Central Nervous System Administration for Vector Mediated Gene Replacement in a Severe Model of Spinal Muscular Atrophy
Jacqueline J. Glascock,1 Katie L. George,2 Christian L. Lorson.2 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO; 2Veterinary Pathobiology, University of Missouri, Columbia, MO; 3Veterinary Pathobiology, University of Missouri, Columbia, MO. 1
Spinal Muscular Atrophy, an autosomal recessive neurodegenerative disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). In humans, a nearly identical copy gene is present, SMN2. SMN2 is retained in all SMA patients, and encodes the same protein as SMN1. However, SMN1 and SMN2 differ by a silent C to T transition at the 5’ end of exon 7, causing alternative splicing of SMN2 transcripts and low levels of full-length SMN. SMA is monogenic and therefore well suited for gene replacement strategies. Recently, self-complementary AAV vectors have been successfully used to deliver the SMN cDNA to an animal model of disease, the SMNΔ7 mouse. scAAV9-SMN and splice-switching oligonucleotides have shown tremendous promise when delivered to the periphery; however, little therapeutic benefit was derived if delivered beyond P4. In this study we examined the extent S70
of the therapeutic window in SMA mice by delivering vector at P2 – P8. Additionally, we wished to explore if post-symptomatic rescue of the SMNΔ7 model if achievable. Utilizing intracerebroventricular injections we delivered scAAV9-SMN on post natal days 2 through 8 and monitored the animals for phenotypic improvement. We observed that injections at earlier time points resulted in greater phenotypic improvement than those at later time points, but that even with day 7 delivery, there was a statistically significant extension in survival. This in contrast to the delivery of vector using intravenous injections in which minimal therapeutic benefit can be detected if delivered at or beyond P4. In the ICV injections, there was a decrease in the degree of rescuing seen with each successive day in which the vector was delivered regarding body weight and survival.
However, vector administration at each time point (P2-P7) proved to be significantly beneficial in extending lifespan and improving body mass. These results indicate that while early pre-symptomatic scAAV delivery is the most beneficial, post-symptomatic delivery directly to the central nervous system can still provide a significant benefit to this important model of SMA.
176. AAV-Mediated Gene Therapy in Limb-Girdle Muscular Dystrophy 2I (LGMD2I) Mouse Model
Chi-Hsien Wang,1 Bin Xiao,1 Chungping Qiao,1 Yiumo M. Chan,2 Ru-Hang Tang,1 Juan Li,1 Elizabeth Keramaris-Vrantsis,2 Peijuan Lu,2 Qilong Lu,2 Xiao Xiao.1 1 Department of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel Hill, NC; 2McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC.
Fukutin-related protein (FKRP) is a putative glycosyltransferase. The genetic mutations of the FKRP cause a wide spectrum of disease phenotypes ranging from late onset and milder limb-girdle muscular dystrophy 2I (LGMD2I) without central nervous system defect to severe allelic diseases such as congenital muscular dystrophy 1C Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
Yi Lai,1 Junling Zhao,1 Yongping Yue,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO.
1
Sarcolemmal neuronal nitric oxide synthase (nNOS) is crucial for normal muscle function. Absence of sarcolemmal nNOS plays a critical role in the pathogenesis of muscular dystrophy. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Dystrophin anchors nNOS to the sarcolemma. Utrophin is a homolog of dystrophin and has been extensively investigated as a candidate gene for DMD gene therapy. Unfortunately, unlike dystrophin, utrophin cannot recruit nNOS to the sarcolemma. Inability of utrophin to restore sarcolemmal nNOS may significantly attenuate its therapeutic effect. To overcome this drawback of utrophin, we set to engineer novel utrophin genes that can fix nNOS to the muscle cell membrane. We recently found that dystrophin spectrin-like repeats 16 and 17 (R16/17) mediate sarcolemmal nNOS localization. Utrophin R15/16 shares great homology with utrophin R16/17. In this study, we examined whether replacing utrophin R15, or R16 or both with respective dystrophin R16 and R17 can result in novel utrophin genes capable of nNOS anchoring. A series of four repeats micro-utrophin AAV vectors were generated. All micro-utrophin genes contained a flag tag, utrophin N-terminus, utrophin repeats 1, and 22 and utrophin cysteine-rich domain. The two repeats between utrophin R1 and R22 were either original utrophin R15/16 or engineered with dystrophin R16/17 substitution. The micro-utrophin gene was packaged into AAV-9 and delivered AAV viruses to the tibialis anterior muscle of utrophin and dystrophin double knockout mice. One month after injection, we examined microgene expression and sarcolemmal nNOS localization. Robust expression was observed in all AAV infected muscle. As expected, the microgene construct carrying original utrophin R15/16 did not recover sarcolemmal nNOS. Constructs with either dystrophin R16 or R17 failed to restore nNOS to the sarcolemma. However when both repeats were replaced, sarcolemmal nNOS expression was restored. Our results suggest that both dystrophin R16 and R17 are required for nNOS localization. Further, dystrophin R16/17 is sufficient for sarcolemmal nNOS expression in the context of utrophin. The novel micro-utrophin gene described here may hold great promise for DMD gene therapy.
175. Determining the Therapeutic Window: Central Nervous System Administration for Vector Mediated Gene Replacement in a Severe Model of Spinal Muscular Atrophy
Jacqueline J. Glascock,1 Katie L. George,2 Christian L. Lorson.2 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO; 2Veterinary Pathobiology, University of Missouri, Columbia, MO; 3Veterinary Pathobiology, University of Missouri, Columbia, MO. 1
Spinal Muscular Atrophy, an autosomal recessive neurodegenerative disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). In humans, a nearly identical copy gene is present, SMN2. SMN2 is retained in all SMA patients, and encodes the same protein as SMN1. However, SMN1 and SMN2 differ by a silent C to T transition at the 5’ end of exon 7, causing alternative splicing of SMN2 transcripts and low levels of full-length SMN. SMA is monogenic and therefore well suited for gene replacement strategies. Recently, self-complementary AAV vectors have been successfully used to deliver the SMN cDNA to an animal model of disease, the SMNΔ7 mouse. scAAV9-SMN and splice-switching oligonucleotides have shown tremendous promise when delivered to the periphery; however, little therapeutic benefit was derived if delivered beyond P4. In this study we examined the extent S70
of the therapeutic window in SMA mice by delivering vector at P2 – P8. Additionally, we wished to explore if post-symptomatic rescue of the SMNΔ7 model if achievable. Utilizing intracerebroventricular injections we delivered scAAV9-SMN on post natal days 2 through 8 and monitored the animals for phenotypic improvement. We observed that injections at earlier time points resulted in greater phenotypic improvement than those at later time points, but that even with day 7 delivery, there was a statistically significant extension in survival. This in contrast to the delivery of vector using intravenous injections in which minimal therapeutic benefit can be detected if delivered at or beyond P4. In the ICV injections, there was a decrease in the degree of rescuing seen with each successive day in which the vector was delivered regarding body weight and survival.
However, vector administration at each time point (P2-P7) proved to be significantly beneficial in extending lifespan and improving body mass. These results indicate that while early pre-symptomatic scAAV delivery is the most beneficial, post-symptomatic delivery directly to the central nervous system can still provide a significant benefit to this important model of SMA.
176. AAV-Mediated Gene Therapy in Limb-Girdle Muscular Dystrophy 2I (LGMD2I) Mouse Model
Chi-Hsien Wang,1 Bin Xiao,1 Chungping Qiao,1 Yiumo M. Chan,2 Ru-Hang Tang,1 Juan Li,1 Elizabeth Keramaris-Vrantsis,2 Peijuan Lu,2 Qilong Lu,2 Xiao Xiao.1 1 Department of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel Hill, NC; 2McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC.
Fukutin-related protein (FKRP) is a putative glycosyltransferase. The genetic mutations of the FKRP cause a wide spectrum of disease phenotypes ranging from late onset and milder limb-girdle muscular dystrophy 2I (LGMD2I) without central nervous system defect to severe allelic diseases such as congenital muscular dystrophy 1C Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy