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Query: UMLS:C0001486 (Adenovirus)
 3,125 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of approximately 8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (approximately 14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus packaging signals, a beta-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1 + E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (> 10(8)/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues.
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PMID:Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells. 881 25

Adenovirus (AV)-mediated gene transfer into skeletal muscles of adult immune-competent animals has been limited by the fact that a cell-mediated immune attack of the host against transduced muscle fibers prevented efficient long-term transgene expression. More recently, various immunomodulating strategies have been shown to improve the longevity of transgene expression after AV-mediated gene transfer. In this study we treated adult dystrophic (mdx) mice with daily subcutaneous injections of the immunosuppressive drug FK506 (tacrolimus) over 5, 10, 30 and 60 days after AV-mediated dystrophin gene transfer and compared the transduction level with saline-injected mdx controls. We show that daily FK506 treatment after AV-mediated dystrophin gene transfer into adult mdx muscle results in the maintenance of the initial transgene expression for at least 2 months, even when FK506 treatment was discontinued after 1 month. This is in keeping with the marked reduction of inflammatory infiltrates and the reduced activation level (inducible nitric oxide synthase) of macrophages in adenoviral recombinant (AVR)-injected muscles of FK506-treated animals. Moreover, we find that FK506 efficiently suppresses the humoral immune response against both the vector proteins and the transgene protein product (dystrophin). Furthermore, we demonstrate that continuous FK506 treatment over 30 days significantly improves the efficiency of gene transfer when the same vector is readministered to an animal which had been transduced 20 days earlier. In conclusion, the data suggest that sensitization by the initial antigenic load of the AVR application plays a pivotal role in triggering the humoral and cellular immune response of the host, which can be significantly counteracted by relatively short-term immunosuppressive treatment. These findings have important implications for the design of future human trials for gene replacement therapy in Duchenne muscular dystrophy.
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PMID:Transient immunosuppression by FK506 permits a sustained high-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscles of adult dystrophic (mdx) mice. 885 96

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes).
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PMID:Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system. 1876 28

Chromosomal correction of dystrophin gene mutations is a most desirable therapeutic solution for Duchenne muscular dystrophy, as it allows production of the full-length dystrophin under the control of locus-specific promoters. Here we explored gene targeting in conditionally immortal mouse dystrophin-deficient myoblasts. We constructed an adenoviral vector for the correction of the mdx mutation, containing 6.0 kb of sequence homologous to the target locus (partial intron 21 through to exon 24 with the normal sequence of exon 23) and a neomycin expression cassette inserted in intron 23. Adenovirus-based gene targeting was previously reported to be beneficial in mouse embryonic stem cells, resulting in one targeted integration per three integration events. However, we found no targeted integration events among 144 stably transduced G418-resistant myoblast clones, reflecting efficient random integration of the adenoviral vector in myogenic cells. We found that mouse myoblasts are capable of integrating recombinant adenoviral DNA with an efficiency approaching 1%. Interestingly, dermal fibroblasts integrate adenoviral DNA up to 100 times less efficiently than myoblasts from the same mice. We also show that the efficiency of recombinant adenoviral DNA integration is influenced by preinfection cell density, possibly indicating the importance of cellular DNA replication for adenoviral integration.
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PMID:Adenovirus-based targeting in myoblasts is hampered by nonhomologous vector integration. 1878 4

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD.
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PMID:CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice. 2695 18