Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The best way to overcome immunorejection in heterologous myoblast transfer (HMT) is by the use of immunodeficient and/or highly immunosuppressed mice as hosts. The same may be attained by autologous myoblast transfer (AMT). In this paper, we describe myoblast transfer in mdx and normal mice where the donor myogenic cells originated from highly inbred litter mates that are considered to be isogenic and thus the procedure is analogous to AMT. The myoblasts were marked in vitro with Rous Sarcoma Virus (RSV)-luciferase (Lux) or RSV-beta-galactosidase (LacZ) reporter genes through transduction mediated by an autonomously replication-defective recombinant human adenovirus. This permitted us to follow their fate after transplantation. mdx and normal mice were irradiated with 20 Gray gamma rays; necrosis and regeneration were induced by intramuscular notexin prior to myoblast injection. In both mdx and normal mice, the expression of luciferase rapidly declined after the injection implying that a large portion of the injected myoblasts were lost by 48 hr, due to undetermined cause(s). The surviving, injected myoblasts well-mosaicized large groups of host fibers but only in the immediate vicinity of the injection. Substantial expression of the reporter gene continued up to 1 month post-transplantation in normal mice, but there was a gradual decline and eventual disappearance of the reporter gene expression in mdx mice. This latter phenomenon was due to the ongoing intense necrosis of muscle fibers in mdx. There was no evidence of immunorejection. These experiments indicate that even in the absence of immunorejection, myoblast transfer suffers from important negative features: major loss of myoblasts within 48 hr after the injection and lack of significant spread of the injected cells from the injection site in the host muscle. These factors, plus the limited proliferative and fusion capacity of Duchenne muscular dystrophy (DMD) myoblasts, make them less than an ideal vector for the dystrophin cDNA for dystrophin gene replacement therapy in DMD.
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PMID:Gene transfer into skeletal muscles by isogenic myoblasts. 794 44

A possible treatment for Duchenne muscular dystrophy is the injection of normal myoblasts into dystrophic muscles to induce the formation of new, healthy, and dystrophin-positive muscle fibers. To develop this therapy, it is important to identify the muscle fibers formed by the injected myoblasts in the host muscles. In this study, we used myoblasts from transgenic mice which have a gene expressing beta-galactosidase under the control of the promoter of quail fast skeletal muscle troponin I. This transgene is expressed in myotubes and muscle fibers, but not in myoblasts. Twenty-eight days after myoblast transplantation in nude and in mdx mice, muscle fibers containing of beta-galactosidase were identified by x-gal staining. In mdx mice, most of the beta-galactosidase-positive muscle fibers resulting from the myoblast transplantation were also dystrophin positive. This technique could make it possible to follow the success of myoblast transplantation even in mice that are not depleted of dystrophin.
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PMID:Utilization of myoblasts from transgenic mice to evaluate the efficacy of myoblast transplantation. 806 99

We have compared the efficiency of direct gene transfer in normal and regenerating rat skeletal muscle. Muscle necrosis and regeneration was induced by intramuscular injection of bupivacaine in the soleus muscle of adult rats. Plasmids containing beta-galactosidase (beta-gal) or chloramphenicol acetyltransferase (CAT) genes driven by viral promoters were injected 3 days after bupivacaine treatment into the regenerating and the contralateral uninjured muscles. Expression of CAT activity was > 80-fold higher in regenerating compared to control muscles at 7 days post-transfection, but decreased at 30 and 60 days. Southern blot analysis showed that the predominant form of CAT DNA was episomal in transfected muscles; however, CAT activity measurements performed on the same transfected muscles showed no precise correlation between enzymatic activity and amount of plasmid DNA. Expression of beta-gal was detected in numerous regenerating fibers of the injured soleus muscles at 7 days post-transfection; in contrast, only rare positive fibers were found in control muscles. Focal infiltrates of mononuclear cells, which surround and invade selectively beta-gal-positive fiber segments, were observed at 30 days post-transfection, suggesting that immune mechanisms are implicated in the progressive loss of transgenes with time. The finding that regenerating muscle fibers display a higher efficiency of transfection may be relevant to gene therapy of Duchenne muscular dystrophy, because regenerating fibers are numerous in the early stages of the disease.
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PMID:Gene transfer in regenerating muscle. 815 66

Primary human myogenic cells isolated from fetal and adult muscle were infected with a high-titer, Moloney murine leukemia virus (MoMLV)-derived retroviral vector expressing a bacterial beta-galactosidase (beta-gal) gene under long terminal repeat (LTR) control. Gene transfer efficiency averaged 50% in both fetal myoblasts and adult satellite cells, as revealed by beta-gal staining. The reporter gene was stably integrated, faithfully inherited, and expressed at significant levels in myogenic cells for at least 10 generations under clonal growth conditions, and throughout the culture life span upon differentiation into myotubes. Comparable gene transfer efficiency was obtained in myogenic cells from muscle biopsies of patients affected by a number of genetic or acquired myopathies, including Duchenne muscular dystrophy. Transduced normal human satellite cells were injected into regenerating muscle of immunodeficient mice, where they formed new muscle fibers in which the product of the reporter gene was detectable for 2 months after injection. These results show that retroviral vectors can be used to transfer foreign genes with high efficiency into normal or abnormal primary human myogenic cells, leading to stable expression into mature muscle. Satellite cells engineered in this way might represent an effective tool for gene therapy of muscular dystrophies as well as for systemic delivery of recombinant gene products for correction of inherited and acquired disorders. The human-mouse model described here will allow in vivo testing of such gene therapy approaches.
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PMID:Retroviral vector-mediated gene transfer into human primary myogenic cells leads to expression in muscle fibers in vivo. 818 86

Cyclophosphamide immunosuppression does not permit successful myoblast allotransplantation in mouse. Myoblast transplantation is a potential treatment for Duchenne muscular dystrophy. In one clinical trial, Duchenne patients were immunosuppressed with cyclophosphamide. We report here that myoblasts from transgenic mice expressing the beta-galactosidase reporter gene transplanted in mdx mice failed to form new muscle fibres when cyclophosphamide (2 or 10 mg kg-1 per day) was used for immunosuppression. At the lowest dose of cyclophosphamide (2 mg kg-1 per day), some mdx recipient mice formed antibodies against donor myoblasts; however, no humoral immune reaction was observed at the highest dose (10 mg kg-1 per day). The failure of transplantation under cyclophosphamide treatment was attributed to the low immunosuppressive activity at a low dose and to the toxic action of a high dose of this drug. These results could explain the lack of success of myoblast transplantation in a previous clinical trial.
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PMID:Cyclophosphamide immunosuppression does not permit successful myoblast allotransplantation in mouse. 858 Jul 33

Myoblasts were grown from monkey muscle biopsies and infected in vitro with a defective retroviral vector containing a cytoplasmic beta-galactosidase (beta-gal) gene. These myoblasts were then transplanted to 14 different monkeys, 6 of which were immunosuppressed with FK506. Without immunosuppression, only a few myoblasts and myotubes expressing beta-gal were observed 1 week after the transplantation, but no cells expressing beta-gal were observed after 4 weeks. This result was attributed to immune responses since infiltration by CD4+ or CD8+ lymphocytes was abundant 1 week after transplantation but not after 4 weeks. The expression of interleukin 6 (IL-6), interleukin 2 (IL-2), granulocyte/macrophage colony stimulating factor (GM-CSF), transforming growth factor-beta (TGF-beta) and granzyme B mRNAs was increased in the myoblast-injected muscle indicating that the infiltrating lymphocytes were activated. Moreover, antibodies against the donor myoblasts were detected in 3 out of 6 cases. When the monkeys were immunosuppressed with FK506, muscle fibers expressing beta-galactosidase (beta-gal) were present 1, 4 and 12 weeks after the transplantation. There was neither significant infiltration by CD4 or CD8 lymphocytes, nor antibodies detected. The mRNA expression of most cytokines was significantly reduced as compared to the nonimmunosuppressed monkeys. These results indicate that FK506 is effective in controlling short-term immune reactions following myoblast transplantation in monkeys and suggest that it may prove useful for myoblast transplantation in Duchenne Muscular Dystrophy patients.
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PMID:Myoblast transplantation in monkeys: control of immune response by FK506. 864 94

Female mdx/mdx mice were crossed with non-dystrophic transgenic males expressing the beta-galactosidase (beta-gal) gene under a muscle-specific promoter (TnILacZ1/29). All male offspring were mdx mice and about 50% of them also expressed the beta-gal gene. The beta-gal/mdx mice were selected as recipients for the transplantation of myoblasts from non-transgenic normal BALB/c mice. When host muscles were not irradiated before myoblast transplantation, 4.6% of the muscle fibers in host muscles were dystrophin positive 1 month after transplantation. Most of these dystrophin-positive muscle fibers were also beta-gal positive. About one quarter of these fibers are the result of reverse mutations; most of them have, however, been produced by fusion of donor myoblasts with host muscle fibers or with host myoblasts. The virtual absence of beta-gal-negative fibers indicates that there were no exclusively donor-donor fusions. When host muscles were irradiated before myoblast transplantation, roughly the same percentage (5.5%) of dystrophin-positive fibers were formed in the injected muscle, but 42% of them were beta-gal negative. These beta-gal-negative dystrophin-positive muscle fibers were formed by the exclusive fusion of donor myoblasts with one another rather than with host cells. This clearly indicates that myoblast transplantation can form completely new muscle fibers or muscle fiber segments when host satellite cell proliferation is reduced by irradiation. These newly formed muscle fibers had, however, a small diameter and additional myoblast transplantation may be required to increase their size. This situation has some similarities with findings in Duchenne muscular dystrophy patients of more than 6 years of age, who also have a limited proliferation capacity of their satellite cells.
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PMID:Mechanism of increasing dystrophin-positive myofibers by myoblast transplantation: study using mdx/beta-galactosidase transgenic mice. 874 Feb 29

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

Recombinant adenovirus vectors (AdV) hold promise as a means of delivering therapeutic genes to muscle in diseases such as Duchenne muscular dystrophy (DMD). However, we have previously shown that the use of AdV is hampered by the development of reduced force-generating capacity, which occurs within 1 week and is progressive up to at least 1 month after AdV delivery in immune-competent animals. Determinations of muscle force production provide a sensitive and clinically important measure of potential adverse effects of AdV-mediated gene transfer on muscle cell function. In the present study, we investigated the role of AdV-related gene expression and host T lymphocyte responses in the genesis of muscle dysfunction following AdV injection of muscle. We report that UV-irradiation of AdV particles, which reduced AdV transcriptional activity without impairing infectivity (as confirmed by in situ polymerase chain reaction), significantly reversed early (4 days post-injection) AdV-induced contractile impairment in immune-competent mice as well as in mice lacking effective CD8+ T cell activity. The superimposed additional reduction in force-generating capacity normally found between 4 and 30 days post-AdV delivery in immune-competent mice, along with the associated loss of transgene (beta-galactosidase) expression, was largely abrogated by the absence of an intact CD8+ T lymphocyte response. Furthermore, short-term administration of a neutralizing antibody against CD4+ T cells significantly prolonged transgene expression and showed a trend toward mitigation of AdV-induced reductions in force-generating capacity. Cellular infiltration and humoral immune responses against the vector and transgene product were also blunted to varying degrees in the setting of CD8+ or CD4+ T cell deficiency. We conclude that AdV-related gene expression has an early negative (probably toxic) effect on muscle cell function that is independent of CD8+ T cell-mediated immunity. In contrast, further progression of contractile impairment and the accompanying loss of transgene expression from AdV-injected muscle are largely dependent upon the activity of CD8+ T cells. These results have implications for the design of future generation vectors and the potential need for immunosuppressive therapy after AdV-mediated gene transfer to muscle.
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PMID:Impairment of force generation after adenovirus-mediated gene transfer to muscle is alleviated by adenoviral gene inactivation and host CD8+ T cell deficiency. 889 73

Duchenne muscular dystrophy (DMD) is an important target for gene transfer because of the disease's high frequency and devastating course. To date, adenoviral vector-mediated gene transfer for DMD has been unavailable because (1) adenoviral vectors were unable to accommodate the full-length dystrophin cDNA (14 kb); and (2) adenoviral vectors induced inflammatory reactions in the gene transfer recipient. We addressed both problems with a novel adenoviral vector that contains no viral genes and encodes 28.2 kb of foreign DNA including both the full-length dystrophin cDNA with the muscle creatine kinase promoter for transcriptional control and a lacZ marker gene. This report presents the in vivo expression of dystrophin and beta-galactosidase from this vector in skeletal muscle of the mdx mouse, a mutant mouse that lacks dystrophin. Somatic delivery of the vector by intramuscular injection in 6-day-old mice resulted in the expression of full-length, recombinant dystrophin at the muscle membrane. Dystrophin-associated proteins were restored in muscle fibers expressing recombinant dystrophin. Mdx muscle injected with our vector showed a decrease in the proportion of fibers with nuclei located centrally; centrally placed nuclei in muscle fibers are characteristic of cycles of degeneration and regeneration suffered by dystrophin-deficient muscle tissue. These results are strong evidence that adenoviral vector-mediated full-length dystrophin delivery provides substantial somatic function.
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PMID:In vivo muscle gene transfer of full-length dystrophin with an adenoviral vector that lacks all viral genes. 894 Jun 36


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