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William E Grose,
K Reed Clark,
Danielle Griffin,
Vinod Malik, Kimberly M Shontz,
Chrystal L Montgomery,
Sarah Lewis,
Robert H Brown,
Paul M L Janssen,
Jerry R Mendell,
Louise R Rodino-Klapac
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ABSTRACT: The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however 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, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.
PLoS ONE 01/2012; 7(6):e39233. · 4.09 Impact Factor
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Louise R Rodino-Klapac,
Chrystal L Montgomery,
William G Bremer, Kimberly M Shontz,
Vinod Malik,
Nancy Davis,
Spencer Sprinkle,
Katherine J Campbell,
Zarife Sahenk,
K Reed Clark,
Christopher M Walker,
Jerry R Mendell,
Louis G Chicoine
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ABSTRACT: Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50-79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.
Molecular Therapy 11/2009; 18(1):109-17. · 6.87 Impact Factor
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ABSTRACT: We have cloned the swine eNOS promoter and analyzed its function in newborn swine pulmonary artery endothelial cells (PAECs). Analysis of the 2.1 kb 5′ flanking region revealed that the swine eNOS promoter is, like its counterparts in human and other species, a TATA-less promoter. The transcription start site, determined by 5′ RLM-RACE, was located 62 bp upstream of the translation start codon. Promoter activity was demonstrated by transient transfection of 5′ deletion promoter/luciferase constructs into swine PAECs, and indicated that the proximal region from − 227 to − 82 was necessary for basal promoter activity. Positive cis-regulatory elements were present from − 227 to − 1290, while negative cis-regulatory elements may be present from − 1290 to − 1926 bp. Electrophoretic mobility shift assay (EMSA) of the proximal region demonstrated that multiprotein complexes were formed in the conserved proximal region of the swine eNOS promoter and a novel Spl site at − 68/ − 59 was involved in the formation of these complexes.
07/2009; 19(1):62-67.
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ABSTRACT: We have cloned the swine eNOS promoter and analyzed its function in newborn swine pulmonary artery endothelial cells (PAECs). Analysis of the 2.1 kb 5' flanking region revealed that the swine eNOS promoter is, like its counterparts in human and other species, a TATA-less promoter. The transcription start site, determined by 5' RLM-RACE, was located 62 bp upstream of the translation start codon. Promoter activity was demonstrated by transient transfection of 5' deletion promoter/luciferase constructs into swine PAECs, and indicated that the proximal region from -227 to -82 was necessary for basal promoter activity. Positive cis-regulatory elements were present from -227 to -1290, while negative cis-regulatory elements may be present from -1290 to -1926 bp. Electrophoretic mobility shift assay (EMSA) of the proximal region demonstrated that multiprotein complexes were formed in the conserved proximal region of the swine eNOS promoter and a novel Spl site at -68/-59 was involved in the formation of these complexes.
DNA Sequence 06/2007; 19(1):62-7. · 0.75 Impact Factor
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ABSTRACT: The present study evaluated mechanical stretch-induced apoptosis in swine vascular smooth muscle cells (VSMC) of different phenotypes. We demonstrated that differentiated VSMC express a greater level of Bcl-2-associated death factor (BAD) and have a significant cell loss when exposed to mechanical stretch (10% elongation, 1 Hz) for 24 h. We further demonstrated that apoptosis was significantly increased only in differentiated VSMC exposed to mechanical stretch. To test the hypothesis that the intracellular level of BAD in VSMC determines its response to mechanical stretch-induced apoptosis, we examined whether BAD expression was upregulated by mechanical stretch-induced apoptosis and was associated with the increase in the apoptosis level of differentiated VSMC. When exposed to mechanical stretch, the expression of BAD in differentiated VSMC was elevated at 1 h and remained at higher levels during the application of stretch (24 h). In contrast, Bcl-2 expression was suppressed during the application of stretch. Moreover, the proapoptotic function of BAD was inhibited by overexpression of Bcl-2 through transient transfection of VSMC with pCEP4-Bcl-2 or incubation of VSMC with vascular epithelial growth factor. These results suggest that mechanical stretch-induced VSMC apoptosis is phenotype dependent. The higher levels of apoptosis of differentiated VSMC upon mechanical stretch were, at least in part, dependent on their intrinsic level of BAD.
Journal of Vascular Research 02/2006; 43(3):229-37. · 2.65 Impact Factor
