Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We established an efficient method for obtaining expression of a foreign marker gene transferred in vitro into myoblasts and in vivo into adult mouse skeletal muscles using adenovirus vector. After infection of the C2 myoblasts with the adenovirus vector containing the beta-actin promoter with cytomegalovirus (CMV) enhancer (CAG promoter) AxCALacZ, significantly greater number of cells express beta-galactosidase when compared with the adenovirus vector expressing the lacZ gene under the control of the SR alpha viral terminal repeat promoter (AxSRLacZL) or the myosin heavy chain (MHC) IIB promoter (AxMHCLacZ). We also injected AxCALacZ into the skeletal muscles of 5- to 6-week-old C57BL/10 mice and determined that more than 60% of their muscle fibers expressed the lacZ gene 7 days after injection. The CAG promoter may have application in the development of gene therapy for Duchenne muscular dystrophy (DMD) using adenovirus vector.
...
PMID:Effective adenovirus-mediated gene expression in adult murine skeletal muscle. 1033 58

Patterns of dystrophin and beta-galactosidase expression were examined in mdx mice after i.m. injections of synthetic microspheres (MF-2) loaded with full-length (pHSADy) or mini-dystrophin gene (pSG5dys) cDNA plasmid constructs or with LacZ marker gene (pCMV-LacZ). A single injection of 25 microg pHSADy into quadriceps femoris muscle resulted in 6.8% of dystrophin positive myofibers (DPM) in a given muscle; 8.4% of DPM in glutaeus muscle and 4.3% of DPM in quadriceps femoris muscle of contralateral limb on day 21 after exposure compared with only 0.6% DPM in intact (non-injected) mdx mice. A high proportion of DPM (17.6% and 10.8%, respectively) was registered in both injected and contralateral muscles after mini- gene cDNA administration. MF-2/dystrophin cDNA particles were detected by FISH analysis in about 60-70% of myofiber nuclei in muscles of injected and contralateral limbs 7 days after application. The presence of human dystrophin cDNA and its products in all skeletal muscles and in different internal organs was proven by PCR and RT-PCR analysis. Patches of beta-galactosidase expression were abundant in injected muscle, and frequent in the contralateral and other skeletal muscles as well as in diaphragm, heart and lungs. High levels of dystrophin cDNA expression, and an efficient distant transfection effect with preferential intranuclei inclusion of MF-2 vehicle, are very encouraging for the development of a new constructive strategy in gene therapy trials of DMD.
...
PMID:Local and distant transfection of mdx muscle fibers with dystrophin and LacZ genes delivered in vivo by synthetic microspheres. 1046 65

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.
...
PMID:Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice. 1075 47

Transplantation of disaggregated myoblasts from normal donor to the muscles of a diseased host, or reimplantation of genetically modified host myoblasts, has been suggested as a possible route to therapy for inherited myopathies such as Duchenne muscular dystrophy, or to supply missing proteins that are required systemically in diseases such as hemophilia. With two exceptions, studies of myoblast transfer in the mouse have involved transplantation of donor myoblasts isolated from adult or neonatal skeletal muscle satellite cells. In this study we present evidence that thymic myoid cells are capable of participating in the regeneration of postnatal skeletal muscle, resulting in the expression of donor-derived proteins such as dystrophin and retrovirally encoded proteins such as beta-galactosidase within host muscles. This leads us to conclude that thymic myoid cells may provide an alternative to myoblasts derived from skeletal muscle as a source of myogenic cells for myoblast transfer.
...
PMID:Thymic myoid cells as a source of cells for myoblast transfer. 1103 69

Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% beta-galactosidase-positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.
...
PMID:Intraarterial injection of muscle-derived CD34(+)Sca-1(+) stem cells restores dystrophin in mdx mice. 1126 50

Ex vivo gene therapy of Duchenne muscular dystrophy based on autologous transplantation of genetically modified myoblasts is limited by their premature senescence. MyoD-converted fibroblasts represent an alternative source of myogenic cells. In this study the forced MyoD-dependent conversion of murine NIH-3T3 fibroblasts into myoblasts under the control of an inducible promoter silent in the presence of tetracycline was evaluated. After tetracycline withdrawal this promoter drives the transcription of MyoD in the engineered fibroblasts, inducing their myogenesis and giving rise to beta-galactosidase-positive cells. MyoD-expressing fibroblasts withdrew from the cell cycle, but were unable to fuse in vitro into multinucleated myotubes. Five days following implantation of engineered fibroblasts in muscles of C57BL/10J mice we observed a sevenfold increase of beta-galactosidase-positive regenerating myofibers in animals not treated with antibiotic compared with treated animals. After 1 week the number of positive fibers decreased and several apoptotic myonuclei were detected. Three weeks following implantation of MyoD-converted fibroblasts in recipient mice, no positive "blue" fiber was observed. Our results suggest that transactivation by tetracycline of MyoD may drive an in vivo myogenic conversion of NIH-3T3 fibroblasts and that, in this experimental setting, apoptosis plays a relevant role in limiting the efficacy of engineered fibroblast transplantation. This work opens the question whether apoptotic phenomena also play a general role as limiting factors of cell-mediated gene therapy of inherited muscle disorders.
...
PMID:In vitro and in vivo tetracycline-controlled myogenic conversion of NIH-3T3 cells: evidence of programmed cell death after muscle cell transplantation. 1133 36

Recombinant adeno-associated (rAAV) viral vectors hold great therapeutic potential for human diseases. However, a relatively small packaging capacity (less than 5 kb) has limited the application of rAAV for certain diseases such as cystic fibrosis and Duchenne muscular dystrophy. Here we compared two mechanistically distinct approaches to overcome packaging restraints with rAAV vectors. The trans-splicing approach reconstitutes gene expression from two independent rAAV vectors, each encoding unique, nonoverlapping halves of a transgene. This process requires intermolecular concatamerization and subsequent splicing between independent vectors. A distinct overlapping vector approach uses homologous recombination between overlapping regions in two independent vectors. Using the beta-galactosidase gene as template, trans-splicing approaches were threefold (in primary fibroblasts) and 12-fold (in muscle tissue) more effective in generating full-length transgene products than the overlapping vector approach. Nevertheless, the efficiency of trans-splicing remained moderate at approximately 4.3% (for muscle) and 7% (for fibroblasts) of that seen with a single vector encoding the full-length transgene. The efficiency of trans-splicing was augmented 1185-fold by adenoviral E4, but not E2a, gene products. This augmentation was much less pronounced with the overlapping vectoring approach (12-fold). Trans-splicing and overlapping vector approaches are two viable alternatives to expand rAAV packaging capacity.
...
PMID:Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. 1159 43

Muscle tissue from Duchenne muscular dystrophy patients and the Dmd(mdx/mdx) (hereafter referred to as mdx) mouse is characterized by an abundance of necrotic myofibers and infiltrating macrophages. Both features may provide additional stimulus to the immune response directed against novel antigens, such as those delivered by gene therapy vectors. It has previously been shown that the immune evasion achieved by adeno-associated virus in healthy muscle fails in one model of muscular dystrophy. Here, we examined the immune response to adenoviral vectors and their transgenes in normal and mdx mice. We found that mdx mouse muscles contain 20 times more macrophages and 7 times more dendritic cells than healthy muscles. This higher professional antigen-presenting cell content results in a stronger immune response to antigens that can be directly presented by those cells, including viral antigens and constitutively expressed transgene products. However, we did not detect a significant immune response to beta-galactosidase expressed specifically in muscle, even at high expression levels. This result suggests that cross-presentation is not more effective in mdx mouse muscle, and that targeted vectors and tissue-specific promoters may be useful tools for evasion of the immune response in dystrophic muscle.
...
PMID:Immune evasion by muscle-specific gene expression in dystrophic muscle. 1173 36

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.
...
PMID:[Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene]. 1186 12

Duchenne muscular dystrophy (DMD) is an X-linked lethal disorder caused by a defect in the DMD gene, which encodes the cytoskeletal protein dystrophin. Utrophin is an autosomal homolog of the DMD gene product dystrophin, and augmented expression of endogenous utrophin is expected to provide an alternative therapeutic approach to DMD. We previously reported that an immune response against a beta-galactosidase-expressing adenovirus vector, AxCALacZ, resulted in an accumulation of endogenous utrophin on the extrasynaptic sarcolemma in dystrophin-deficient mdx mice. To determine which cytokine is involved in the regulation of utrophin expression, we directly injected several cytokines separately into neonatal mdx muscles and tested whether the expression of utrophin is increased on the sarcolemma. Importantly, among the cytokines tested, solely interleukin 6 (IL-6) successfully increased expression of utrophin. Moreover, the increase in utrophin mRNA was detected in recombinant IL-6-injected mdx muscles by quantitative real-time reverse transcriptase-polymerase chain reaction. Further, IL-6 expression was elevated in AxCALacZ-infected mdx muscle at an early stage, and anti-IL-6 receptor (IL-6R) antibody treatment blocked enhanced utrophin expression in AxCALacZ-infected mdx muscle. We should point out, however, that overexpression of utrophin due to recombinant IL-6 treatment lasted only 1 week. In addition, expression of utrophin was not evident in normal C57BL/10 neonatal muscles injected with IL-6. Taken together, these results suggest that IL-6 can induce overexpression of utrophin on the extrasynaptic sarcolemma but requires preexisting factors in neonatal mdx muscle to fully regulate utrophin expression.
...
PMID:Interleukin 6 induces overexpression of the sarcolemmal utrophin in neonatal mdx skeletal muscle. 1187 29


<< Previous 1 2 3 4 5 Next >>

---