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616. Transfection of Murine Dermis Via Topical DNA Plasmid Delivery
MUSCULO-SKELETAL GENE & CELL THERAPY II 615. The Phenotype of Dysferlin-Deficient Mice Is Not Rescued by AAV-Mediated Transfer of Anoctamin 5
Karine Charton,1 Laurence Suel-Petat,1 François Monjaret,1 Nathalie Bourg-Alibert,1 Carinne Roudaut,1 Evelyne Gicquel,1 Bjarne Udd,2 Isabelle Richard.1 1 Genethon, EVRY, France, Metropolitan; 2Neuromuscular Research Unit, Tampere, Finland.
Mutations in dysferlin and anoctamin 5 have been shown to lead to Limb-girdle Muscular Dystrophy type 2B and 2L respectively and also lead both to Miyoshi Myopathy. These two proteins have been both implicated in sarcolemmal resealing. On the basis of similarities in associated phenotypes and protein functions, we tested the hypothesis that ANO5 protein could compensate for dysferlin deficiency. We first defined that the main transcript of ANO5 expressed in human skeletal muscle is the 22-exon full-length isoform and we demonstrated that dysferlin deficient mice (Dysfprmd) have lower ANO5 expression levels, observation that further enhanced the rational of the tested hypothesis. We constructed an Adeno-Associated Virus (AAV) vector that encoded for human ANO5 and performed intramuscular injection in wild-type (WT) and dysferlin deficient mice. We showed that AAVmediated transfer of ANO5 did not lead to apparent toxicity in WT mice. We then demonstrated that AAV-hANO5 injection does not improve the membrane repair defect seen in the absence of dysferlin as tested by laser-wounding. In addition, ANO5 overexpression did not improve muscle histology or prevented damage to muscle following eccentric exercise in vivo in the dysferlin deficient model. Consequently, overexpressing hANO5 does not seem to provide a valuable therapeutic strategy for dysferlin deficiency.
616. Transfection of Murine Dermis Via Topical DNA Plasmid Delivery
Chunqing Dou,1 Guy P. Marti,1 Lixin Liu,1 Frank Lay,1 Donald J. Rees,1 Aaron T. Tabor,2 Junkai Du,1 Sayed Mohammd Hosseini,1 Amir Mehdi Ansari,1 Ali Karim Ahmed,1 James A. Williams,3 John W. Harmon.1 1 Hendrix Burn/Wound Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD; 2Gene Facelift, Winston Salem, NC; 3R&D, Nature Technology Corporation, Lincoln, NE. Techniques for topical gene delivery to the skin reported so far have required chemical alteration of the skin barrier or transport particles (liposomes or chitosan/tripolyphosphate [TPP] nanoparticles) and are not exempt of safety concerns. Here we present a novel approach that permits gene transfer and expression following topical application of naked plasmid DNA to mouse skin without physically or chemically aggressive means. NTC8385-VA1 (Natural Technology Corp. Lincoln, NE), a potent minimalized antibiotic-free plasmid vector with VA1 and HTLV-1 R expression enhancers, was used to transfer the reporter gene Luciferase and target gene KGF. Twenty 10-week old male SV129 mice were randomly and evenly assigned to Luciferase and KGF groups. The dorsum was shaved with hair clipper and depilated by Nair*. The microdermabrasion zone was brushed firmly with a coarse tip of a Dermasweep-Mini machine (Dermasweep, Rocklin, CA) at 20mmHg pressure. Fifty mg NTC8385-VA1-Luciferase plasmid or NTC8385-VA1-KGF plasmid diluted in methylcellulose was topically applied and then covered with DuoDERM* on each brushed zone every 12 hours. Luciferase imaging was used to assess transfection efficiency with KGF plasmid as the “empty vector” control. Meanwhile, the biological effect of KGF transfection was assessed, with the Luciferase treated microdermabrasion zone as the negative controls. Mice in the Luciferase group showed significantly increased expression of luciferase at 36, 60, 72 and 96 hours after microdermabrasion. The epithelial thickness in the microdermabrasion zone in the KGF Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
group was significantly increased to 25.9mm in comparison with the Luciferase group, 15.9mm at 48 hours (p = 0.045). Dermal thickness tended to be increased in the KGF group 254.9 vs. 162.1mm at 120 hours (p = 0.057). These findings indicate that plasmid vector NTC8385-VA1 can efficiently deliver target genes into mouse skin with multiple topical applications. Topical application of KGF may be a useful therapeutic to restore atrophic dermis. Key words: topical DNA application, gene therapy, DNA plasmid vector, KGF, dermis, microdermabrasion.
617. Clinical Trial Readiness for Gene Replacement to the Diaphragm: Translational Respiratory Endpoints in Ventilator-Dependent Neuromuscular Diseases
Barbara K. Smith,1 Melissa A. Goddard,2 Martin K. Childers,3 Barry J. Byrne.4 1 Physical Therapy, University of Florida, Gainesville, FL; 2Wake Forest University, Winston-Salem, NC; 3Rehabilitation Medicine and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA; 4Department of Pediatrics and Powell Gene Therapy Center, University of Florida, Gainesville, FL. X-linked myotubular myopathy (XLMTM) is a congenital myopathy characterized by widespread hypotonia, muscle weakness and early mechanical ventilator (MV) dependence of affected boys. Although there is no current treatment for XLMTM, preclinical work in a canine model of XLMTM indicates that gene replacement therapy to the diaphragm could provide a therapeutic option in the future. However, little is known regarding the respiratory muscle function of medically stable, MV-dependent children with XLMTM. Respiratory muscle function has been better characterized in Pompe disease, an inherited neuromuscular disorder also notable for ventilatory failure in early childhood and the focus of an ongoing clinical trial of gene therapy to the diaphragm. We hypothesized that the respiratory muscle outcomes used in Pompe disease could also be used characterize respiratory muscle function in boys with XLMTM. MV-dependent boys with XLMTM (n=1, age 11) and Pompe disease (n=2, ages 9 and 15) underwent respiratory muscle strength and functional tests, and the results were compared to values from unaffected children (n=2, ages 9-10). Despite MV dependence since birth, the XLMTM child was able to spontaneously initiate inspiratory efforts and complete all of the tests. At a comparable age and duration of MV dependence (pooled average 11.3 years), XLMTM and Pompe children experienced an appreciable decline in maximal inspiratory pressure (-93% vs -98%), maximal voluntary ventilation (-92% vs -89%), and best-effort tidal volume (-99% vs -98%), when compared to the performance of the unaffected children. Inspiratory volume and flow compensatory responses were comparable in both neuromuscular diseases, but the ventilatory kinematics during loaded breathing differed between Pompe and XLMTM. We conclude that inspiratory drive may be preserved in some MV-dependent children with XLMTM and a similar battery of tests can produce consistent ventilatory responses. Kinematic differences between the neuromuscular conditions suggest distinctive patterns and degrees of extradiaphragmatic respiratory muscular involvement.
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Karine Charton,1 Laurence Suel-Petat,1 François Monjaret,1 Nathalie Bourg-Alibert,1 Carinne Roudaut,1 Evelyne Gicquel,1 Bjarne Udd,2 Isabelle Richard.1 1 Genethon, EVRY, France, Metropolitan; 2Neuromuscular Research Unit, Tampere, Finland.
Mutations in dysferlin and anoctamin 5 have been shown to lead to Limb-girdle Muscular Dystrophy type 2B and 2L respectively and also lead both to Miyoshi Myopathy. These two proteins have been both implicated in sarcolemmal resealing. On the basis of similarities in associated phenotypes and protein functions, we tested the hypothesis that ANO5 protein could compensate for dysferlin deficiency. We first defined that the main transcript of ANO5 expressed in human skeletal muscle is the 22-exon full-length isoform and we demonstrated that dysferlin deficient mice (Dysfprmd) have lower ANO5 expression levels, observation that further enhanced the rational of the tested hypothesis. We constructed an Adeno-Associated Virus (AAV) vector that encoded for human ANO5 and performed intramuscular injection in wild-type (WT) and dysferlin deficient mice. We showed that AAVmediated transfer of ANO5 did not lead to apparent toxicity in WT mice. We then demonstrated that AAV-hANO5 injection does not improve the membrane repair defect seen in the absence of dysferlin as tested by laser-wounding. In addition, ANO5 overexpression did not improve muscle histology or prevented damage to muscle following eccentric exercise in vivo in the dysferlin deficient model. Consequently, overexpressing hANO5 does not seem to provide a valuable therapeutic strategy for dysferlin deficiency.
616. Transfection of Murine Dermis Via Topical DNA Plasmid Delivery
Chunqing Dou,1 Guy P. Marti,1 Lixin Liu,1 Frank Lay,1 Donald J. Rees,1 Aaron T. Tabor,2 Junkai Du,1 Sayed Mohammd Hosseini,1 Amir Mehdi Ansari,1 Ali Karim Ahmed,1 James A. Williams,3 John W. Harmon.1 1 Hendrix Burn/Wound Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD; 2Gene Facelift, Winston Salem, NC; 3R&D, Nature Technology Corporation, Lincoln, NE. Techniques for topical gene delivery to the skin reported so far have required chemical alteration of the skin barrier or transport particles (liposomes or chitosan/tripolyphosphate [TPP] nanoparticles) and are not exempt of safety concerns. Here we present a novel approach that permits gene transfer and expression following topical application of naked plasmid DNA to mouse skin without physically or chemically aggressive means. NTC8385-VA1 (Natural Technology Corp. Lincoln, NE), a potent minimalized antibiotic-free plasmid vector with VA1 and HTLV-1 R expression enhancers, was used to transfer the reporter gene Luciferase and target gene KGF. Twenty 10-week old male SV129 mice were randomly and evenly assigned to Luciferase and KGF groups. The dorsum was shaved with hair clipper and depilated by Nair*. The microdermabrasion zone was brushed firmly with a coarse tip of a Dermasweep-Mini machine (Dermasweep, Rocklin, CA) at 20mmHg pressure. Fifty mg NTC8385-VA1-Luciferase plasmid or NTC8385-VA1-KGF plasmid diluted in methylcellulose was topically applied and then covered with DuoDERM* on each brushed zone every 12 hours. Luciferase imaging was used to assess transfection efficiency with KGF plasmid as the “empty vector” control. Meanwhile, the biological effect of KGF transfection was assessed, with the Luciferase treated microdermabrasion zone as the negative controls. Mice in the Luciferase group showed significantly increased expression of luciferase at 36, 60, 72 and 96 hours after microdermabrasion. The epithelial thickness in the microdermabrasion zone in the KGF Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
group was significantly increased to 25.9mm in comparison with the Luciferase group, 15.9mm at 48 hours (p = 0.045). Dermal thickness tended to be increased in the KGF group 254.9 vs. 162.1mm at 120 hours (p = 0.057). These findings indicate that plasmid vector NTC8385-VA1 can efficiently deliver target genes into mouse skin with multiple topical applications. Topical application of KGF may be a useful therapeutic to restore atrophic dermis. Key words: topical DNA application, gene therapy, DNA plasmid vector, KGF, dermis, microdermabrasion.
617. Clinical Trial Readiness for Gene Replacement to the Diaphragm: Translational Respiratory Endpoints in Ventilator-Dependent Neuromuscular Diseases
Barbara K. Smith,1 Melissa A. Goddard,2 Martin K. Childers,3 Barry J. Byrne.4 1 Physical Therapy, University of Florida, Gainesville, FL; 2Wake Forest University, Winston-Salem, NC; 3Rehabilitation Medicine and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA; 4Department of Pediatrics and Powell Gene Therapy Center, University of Florida, Gainesville, FL. X-linked myotubular myopathy (XLMTM) is a congenital myopathy characterized by widespread hypotonia, muscle weakness and early mechanical ventilator (MV) dependence of affected boys. Although there is no current treatment for XLMTM, preclinical work in a canine model of XLMTM indicates that gene replacement therapy to the diaphragm could provide a therapeutic option in the future. However, little is known regarding the respiratory muscle function of medically stable, MV-dependent children with XLMTM. Respiratory muscle function has been better characterized in Pompe disease, an inherited neuromuscular disorder also notable for ventilatory failure in early childhood and the focus of an ongoing clinical trial of gene therapy to the diaphragm. We hypothesized that the respiratory muscle outcomes used in Pompe disease could also be used characterize respiratory muscle function in boys with XLMTM. MV-dependent boys with XLMTM (n=1, age 11) and Pompe disease (n=2, ages 9 and 15) underwent respiratory muscle strength and functional tests, and the results were compared to values from unaffected children (n=2, ages 9-10). Despite MV dependence since birth, the XLMTM child was able to spontaneously initiate inspiratory efforts and complete all of the tests. At a comparable age and duration of MV dependence (pooled average 11.3 years), XLMTM and Pompe children experienced an appreciable decline in maximal inspiratory pressure (-93% vs -98%), maximal voluntary ventilation (-92% vs -89%), and best-effort tidal volume (-99% vs -98%), when compared to the performance of the unaffected children. Inspiratory volume and flow compensatory responses were comparable in both neuromuscular diseases, but the ventilatory kinematics during loaded breathing differed between Pompe and XLMTM. We conclude that inspiratory drive may be preserved in some MV-dependent children with XLMTM and a similar battery of tests can produce consistent ventilatory responses. Kinematic differences between the neuromuscular conditions suggest distinctive patterns and degrees of extradiaphragmatic respiratory muscular involvement.
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