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909. Comparison of Early Stage Healing Responses of Injured Flexor Tendons with Tissue Reactions after Delivery of Adenoviral and Adeno-Associated Viral Vectors to Digital Flexor Tendons
MUSCLE AND CONNECTIVE TISSUE II have been produced at titers>10E12 vg/mL and are currently screened utilising local and systemic routes of administration in preclinical models of the disease.
908. Design and Optimization of U7snRNAs for Skipping of Exon 51 in DMD: Promising Tools for Future Clinical Trials Aurelie Goyenvalle,1 Adeline Vulin,1 Stephanie Lorain,1 Annemieke Aartsma-Rus,2 Judith C. T. van Deutekom,2 Olivier Danos,1 Luis Garcia.1 1 DMD Laboratory, Genethon, Evry, France, Metropolitan; 2 Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands. Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion of a single exon can restore the reading frame, given rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). One potential treatment of the disorder has utilized antisense oligoribonucleotide (AO) to induce removal of disease-associated exons during pre-mRNA processing. Indeed, this approach has been successfully used in DMD cells in vitro with antisense sequences against splice junctions of exon 51. Skipping of this exon would theoretically restore a functional quasidystrophin in a significant subset of DMD patients with ∆45-50,∆4750,∆48-50, ∆49-50, ∆50 and ∆52 genotypes. However, since the AO are not self-renewed, they can not achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and lentiviral vectors. We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying antisense sequences against exon 51 of the human dystrophin gene (U7-ex51). Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts, whereas AAV vectors were injected in the transgenic hDMD mice (carrying the human dystrophin gene). We confirmed the skipping of the exon 51 in vitro in human myoblasts after transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also detected the skipping of the exon 51 after intramuscular injection of an AAV-U7ex51 vector in the transgenic hDMD mouse. We have also tested the efficacy of these vectors to restore dystrophin expression in myoblasts from patients with ∆49-50 and ∆52 deletions. In this study, we provide evidence that efficient skipping of exon 51 can be achieved in human cells and also in vivo after intramuscular injection in a transgenic hDMD mice through U7snRNA shuttle. These results offer very promising tools for clinical treatment of DMD.
909. Comparison of Early Stage Healing Responses of Injured Flexor Tendons with Tissue Reactions after Delivery of Adenoviral and AdenoAssociated Viral Vectors to Digital Flexor Tendons Bei Zhu,1 Yi Cao,1 Ke-Qin Xin,3 Ian C Summerhayes,4 Xiao Tian Wang,2 Paul Y. Liu,2 Jin Bo Tang.1,2 1 Department of Hand Surgery, Affiliated Hospital of Nantong University, Nantong, China; 2Surgical Research and Gene Therapy, Roger Williams Medical Center, Providence, RI; 3 Yokohama City University, Yokohama, Japan; 4Cell and Molecular Biology, Lahey Clinic Medical Center, Burlington, MA. PURPOSE Delivery of growth factors that may substantially increase the healing rate of injured digital flexor tendons is a new arena of application of gene therapy. So far adenoviral, adenoS350
associated viral (AAV), and liposome-mediated plasmid vectors have been used to deliver growth factor genes to tendons or tenocytes, but tissue reactions of these vectors in tendons, in particular those in contrast of healing responses of the injured tendons, were unknown. The purpose of this study was to compare tissue reactions of these vectors in tendons and those of healing responses of injured flexor tendons. METHODS Flexor digitorum profundus tendons within sheath area of paws of 4 New Zealand white rabbits were used as a model. Adenoviral vector (Ad5-lacZ), 1 × 1010 viral particles, was injected into each of 9 tendons, and AAV2-luciferase, 1 × 1010 viral particles, was injected into each of 9 tendons. pCMV-b mixed with 3.5 µL of lipofectamine was injected into each tendon. In other 9 tendons, the tendons were cut through volar 2/3 transversely and repaired. At 3, 7, and 14 days, the tendons were harvested and stained with hematoxylin and eosin. Normal tendons were harvested, serving as control. RESULTS Tissue reactions of the liposomeplasmid vectors in tendons were the most prominent among 3 vectors tested. The adenoviral and AAV2 vectors showed similar tissue reactions at the same viral titers tested, but AAV2 had less obvious tissue reactions in the endotenon area than adenoviral vectors. Early stage inflammatory changes were remarkable in the injured tendons. Compared with these tissue reactions, the reactions elicited by the vectors were less severe. CONCLUSIONS The tested 3 gene delivery systems had less severe tissue reactions in flexor tendons compared with inflammatory changes in injured flexor tendons in early stage. Among 3 vector systems tested, adenoviral and AAV vectors elicited less tissue reactions than liposome-plasmid vectors. AAV2 causes less severe reactions in the endotenon area than adenoviral vectors. From the perspective of tissue reactions, adenoviral and AAV2 vectors, in particular AAV2, are advantageous gene delivery systems for future investigation of delivery of genes to promote flexor tendon in vivo.
910. Gene Therapy of Glycogenosis Type 2 Using SIN-Lentiviral Vectors Richard Emmanuel,1 Benhassine Safa,1 Douillard Gaelle,1 Batista Lionel,1 Caillaud Catherine.1 1 Departement Genetique Developpement, Institut Cochin, INSERM U567, Paris, France. Glycogenosis type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by a deficiency of lysosomal acid aglucosidase (GAA), responsible for a glycogen accumulation mainly in skeletal muscle, heart and diaphragm. Two main clinical presentations can be distinguished: the infantile form presenting with severe cardiomyopathy and hypotonia and the adult form exhibiting progressive proximal muscle weakness and impairment of respiratory function. Different therapeutic approaches are currently under development for GSDII patients such as enzyme replacement therapy (ERT) and gene therapy. In this study we aimed to develop a new gene transfer approach based on lentiviral vectors. We first designed SIN-lentiviral vectors expressing GAA or EGFP genes under the control of the ubiquitous retroviral MNDpromoter. Batches of high-titer recombinant lentiviral vectors were obtained after concentration reaching 5.109 IU/ml. Fibroblasts from 4 GSDII patients (2 infantile and 2 adult forms of the disease) were transduced at various multiplicity of infection (MOI 1, 10 and 100). Transduction efficiency based on EGFP-expression analysis ranged from 3 to 93% EGFP+ cells . After GAA gene transfer a strong increase of GAA activity was observed (up to 85 fold vs normal) with a complete restoration obtained at MOI 10 (24 to 34% EGFP+ cells). A slight decrease of glycogen accumulation was observed after GAA gene transfer but only at MOI 100. We then developed lentiviral vectors expressing EGFP or GAA genes under the control of muscle-specific (MS) enhancer/promoters. We compared both the mouse muscle creatin kinase (MCK) minimal promoter (Mp) Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
908. Design and Optimization of U7snRNAs for Skipping of Exon 51 in DMD: Promising Tools for Future Clinical Trials Aurelie Goyenvalle,1 Adeline Vulin,1 Stephanie Lorain,1 Annemieke Aartsma-Rus,2 Judith C. T. van Deutekom,2 Olivier Danos,1 Luis Garcia.1 1 DMD Laboratory, Genethon, Evry, France, Metropolitan; 2 Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands. Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion of a single exon can restore the reading frame, given rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). One potential treatment of the disorder has utilized antisense oligoribonucleotide (AO) to induce removal of disease-associated exons during pre-mRNA processing. Indeed, this approach has been successfully used in DMD cells in vitro with antisense sequences against splice junctions of exon 51. Skipping of this exon would theoretically restore a functional quasidystrophin in a significant subset of DMD patients with ∆45-50,∆4750,∆48-50, ∆49-50, ∆50 and ∆52 genotypes. However, since the AO are not self-renewed, they can not achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and lentiviral vectors. We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying antisense sequences against exon 51 of the human dystrophin gene (U7-ex51). Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts, whereas AAV vectors were injected in the transgenic hDMD mice (carrying the human dystrophin gene). We confirmed the skipping of the exon 51 in vitro in human myoblasts after transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also detected the skipping of the exon 51 after intramuscular injection of an AAV-U7ex51 vector in the transgenic hDMD mouse. We have also tested the efficacy of these vectors to restore dystrophin expression in myoblasts from patients with ∆49-50 and ∆52 deletions. In this study, we provide evidence that efficient skipping of exon 51 can be achieved in human cells and also in vivo after intramuscular injection in a transgenic hDMD mice through U7snRNA shuttle. These results offer very promising tools for clinical treatment of DMD.
909. Comparison of Early Stage Healing Responses of Injured Flexor Tendons with Tissue Reactions after Delivery of Adenoviral and AdenoAssociated Viral Vectors to Digital Flexor Tendons Bei Zhu,1 Yi Cao,1 Ke-Qin Xin,3 Ian C Summerhayes,4 Xiao Tian Wang,2 Paul Y. Liu,2 Jin Bo Tang.1,2 1 Department of Hand Surgery, Affiliated Hospital of Nantong University, Nantong, China; 2Surgical Research and Gene Therapy, Roger Williams Medical Center, Providence, RI; 3 Yokohama City University, Yokohama, Japan; 4Cell and Molecular Biology, Lahey Clinic Medical Center, Burlington, MA. PURPOSE Delivery of growth factors that may substantially increase the healing rate of injured digital flexor tendons is a new arena of application of gene therapy. So far adenoviral, adenoS350
associated viral (AAV), and liposome-mediated plasmid vectors have been used to deliver growth factor genes to tendons or tenocytes, but tissue reactions of these vectors in tendons, in particular those in contrast of healing responses of the injured tendons, were unknown. The purpose of this study was to compare tissue reactions of these vectors in tendons and those of healing responses of injured flexor tendons. METHODS Flexor digitorum profundus tendons within sheath area of paws of 4 New Zealand white rabbits were used as a model. Adenoviral vector (Ad5-lacZ), 1 × 1010 viral particles, was injected into each of 9 tendons, and AAV2-luciferase, 1 × 1010 viral particles, was injected into each of 9 tendons. pCMV-b mixed with 3.5 µL of lipofectamine was injected into each tendon. In other 9 tendons, the tendons were cut through volar 2/3 transversely and repaired. At 3, 7, and 14 days, the tendons were harvested and stained with hematoxylin and eosin. Normal tendons were harvested, serving as control. RESULTS Tissue reactions of the liposomeplasmid vectors in tendons were the most prominent among 3 vectors tested. The adenoviral and AAV2 vectors showed similar tissue reactions at the same viral titers tested, but AAV2 had less obvious tissue reactions in the endotenon area than adenoviral vectors. Early stage inflammatory changes were remarkable in the injured tendons. Compared with these tissue reactions, the reactions elicited by the vectors were less severe. CONCLUSIONS The tested 3 gene delivery systems had less severe tissue reactions in flexor tendons compared with inflammatory changes in injured flexor tendons in early stage. Among 3 vector systems tested, adenoviral and AAV vectors elicited less tissue reactions than liposome-plasmid vectors. AAV2 causes less severe reactions in the endotenon area than adenoviral vectors. From the perspective of tissue reactions, adenoviral and AAV2 vectors, in particular AAV2, are advantageous gene delivery systems for future investigation of delivery of genes to promote flexor tendon in vivo.
910. Gene Therapy of Glycogenosis Type 2 Using SIN-Lentiviral Vectors Richard Emmanuel,1 Benhassine Safa,1 Douillard Gaelle,1 Batista Lionel,1 Caillaud Catherine.1 1 Departement Genetique Developpement, Institut Cochin, INSERM U567, Paris, France. Glycogenosis type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by a deficiency of lysosomal acid aglucosidase (GAA), responsible for a glycogen accumulation mainly in skeletal muscle, heart and diaphragm. Two main clinical presentations can be distinguished: the infantile form presenting with severe cardiomyopathy and hypotonia and the adult form exhibiting progressive proximal muscle weakness and impairment of respiratory function. Different therapeutic approaches are currently under development for GSDII patients such as enzyme replacement therapy (ERT) and gene therapy. In this study we aimed to develop a new gene transfer approach based on lentiviral vectors. We first designed SIN-lentiviral vectors expressing GAA or EGFP genes under the control of the ubiquitous retroviral MNDpromoter. Batches of high-titer recombinant lentiviral vectors were obtained after concentration reaching 5.109 IU/ml. Fibroblasts from 4 GSDII patients (2 infantile and 2 adult forms of the disease) were transduced at various multiplicity of infection (MOI 1, 10 and 100). Transduction efficiency based on EGFP-expression analysis ranged from 3 to 93% EGFP+ cells . After GAA gene transfer a strong increase of GAA activity was observed (up to 85 fold vs normal) with a complete restoration obtained at MOI 10 (24 to 34% EGFP+ cells). A slight decrease of glycogen accumulation was observed after GAA gene transfer but only at MOI 100. We then developed lentiviral vectors expressing EGFP or GAA genes under the control of muscle-specific (MS) enhancer/promoters. We compared both the mouse muscle creatin kinase (MCK) minimal promoter (Mp) Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy