Genes transferred by retroviral vectors into normal and mutant myoblasts in primary cultures are expressed in myotubes.

Smith, B F; Hoffman, R K; Giger, U; Wolfe, John H.

doi:10.1128/mcb.10.6.3268

Cited by 24 publications

(7 citation statements)

References 5 publications

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“…Two approaches have recently been proposed toward this long-term goal: myoblast transplantation (5) and direct introduction of plasmid DNA into mature muscle, by injection or by particle bombardment (6,19). Although retroviruses have been used for gene transfer in muscle cells in culture (20), they cannot be used on noncycling cells and thus on myofibers. Their use would thus also involve myoblast transplantation (for instance, for gene correction using autologous cells).…”

Section: Discussionmentioning

confidence: 99%

Adenovirus as an expression vector in muscle cells in vivo.

Quantin

Perricaudet

Tajbakhsh

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

276

115

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Attempting gene transfer in muscle raises difficult problems: the nuclei of mature muscle fibers do not undergo division, thus excluding strategies involving replicative integration of exogenous DNA. As adenovirus has been reported to be an efficient vector for the transfer of an enzyme encoding gene in mice, we decided to explore its potential for muscle cells. Advantages of adenovirus vectors are their independence of host cell replication, broad host range, and potential capacity for large foreign DNA inserts. We constructed a recombinant adenovirus containing the fi-galactosidase reporter gene under the control of muscle-specific regulatory sequences. This recombinant virus was able to direct expression of the 3-galactosidase in myotubes in vitro. We report its in vivo expression in mouse muscles up to 75 days after infection. The efficiency and stability of expression we obtained compare very favorably with other strategies proposed for gene or myoblast transfer in muscle in vivo.The eventual correction of muscle diseases, and especially Duchenne muscular dystrophy (DMD), raises very difficult problems. As much as 50% ofDMD cases are sporadic (1) and thus cannot be prevented by current genetic counseling and prenatal diagnosis approaches; this frequency is even higher in mitochondrial myopathies (2). It is thus necessary to actively explore therapeutic strategies. The main problems to overcome are the large number of muscles to be treated and the nondividing nature of mature muscle cells. Moreover, the size of the sequences coding for dystrophin [11,056 base pairs (bp)] is incompatible with most vectors used for gene transfer; in addition, dystrophin is an intracellular protein thought to have a structural role in the muscle cells, whereas most of the current gene therapy approaches concern circulating proteins such as a1-antitrypsin (3), or diseases due to an enzymatic defect, where a very partial restoration of the activity might be sufficient, such as adenosine deaminase (4).Two strategies have been proposed to date: myoblast transplantation (5) and direct DNA injection (6). We decided to explore a third route using adenovirus as a vector for gene expression in muscle cells. Such a vector has already been used to correct postnatally an ornithine transcarbamoylase deficiency in mouse (7,8) 2581The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Discussionmentioning

confidence: 99%

Adenovirus as an expression vector in muscle cells in vivo.

Quantin

Perricaudet

Tajbakhsh

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

276

115

View full text Add to dashboard Cite

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“…Sci. USA 89 (1992) be obtained from a small biopsy of muscle tissue of patients, can be efficiently transduced with retrovirus vectors (19) and well characterized in vitro before being injected into the muscle tissues. This ex vivo approach, therefore, provides a method of efficiently introducing into adult muscles a recombinant gene that can be stably integrated into the genome of muscle cells and expressed for a long time.…”

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confidence: 99%

Expression of human factor IX in mice after injection of genetically modified myoblasts.

Yao

Kurachi

1992

Proc. Natl. Acad. Sci. U.S.A.

142

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Hemophilia B is an X chromosome-linked recessive bleeding disorder. To develop a somatic gene therapy for this disease, we have examined whether mouse skeletal myoblasts can serve as efficient vehicles for systemic delivery of recombinant factor IX. When mouse myoblasts (C2C12) transduced with a Moloney murine leukemia virus-based vector containing the bacterial f-galactosidase gene were i 'ected into mouse skeletal muscles, they fused with the existing and regenerating myofibers and continued to express P-galactosidase. C2C12 myoblasts that were infected with recombinant retroviruses containing a human factor Ia cDNA secreted biologically active human factor IX into the culture medium at a rate of 2.6 zg per 10' cells per day. Myotubes derived from these cells in culture continued to express human factor IX (0.68 itg/day from myotubes derived from 10' C2C12 cells).After injection of the transduced C2C12 myoblasts into skeletal muscles of mice, the systemic level of recombinant human factor IX was found to be as high as -1 ,ug/ml of serum. These results provide the rationale for using skeletal myoblasts as an efficient gene delivery vehicle in the somatic gene therapy for hemophilia B.Factor IX is a plasma glycoprotein that plays a pivotal role in the middle phase of the blood coagulation cascade (1). It is normally synthesized in liver and secreted into the circulation. A deficiency of biologically active factor IX in circulation results in an X chromosome-linked recessive bleeding disorder, hemophilia B (Christmas disease). The current treatment for this disease by plasma protein replacement therapy is effective but complicated by serious side effects, such as possible exposure of patients to blood-borne pathogenic viruses, including hepatitis and human immunodeficiency viruses. Somatic cell gene therapy may provide an alternative safe treatment for this disorder (2). In such an approach, the normal factor IX gene is transferred into target somatic cells that can stably produce active factor IX and transport it into the circulation. The somatic cells used must be able to efficiently carry out various post-translational modifications, such as y-carboxylation, required for the biological activity of factor IX (1). Genetically modified skin fibroblasts implanted in mice as in dermis or subcutaneous implants can produce and secrete recombinant factor IX into the circulation (3, 4). This approach, however, has suffered from poor stability of expression (5) and inefficient transportation of recombinant proteins into the circulation (4).Skeletal myoblasts have several unique properties that make them attractive for use in somatic gene therapy. Proliferating myoblasts are readily isolated and cultured in large numbers (6, 7). More importantly, these cells can fuse with existing muscle fibers when injected into muscle tissues (8).In this report, we demonstrate the expression of the recombinant genes for human factor IX and f3-galactosidase by injecting genetically modified myoblasts into mouse skeletal muscles....

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“…Metabolic Labeling-Primary canine myoblasts were obtained and cultured from a normal and an M-PFK-deficient dog, as described previously (13). The myoblasts were plated at high density (approximately 1.25 ϫ 10 3 /mm 2 ) and allowed to differentiate and form myotubes in Dulbecco's modified Eagle's medium supplemented with 10% horse serum.…”

Section: Methodsmentioning

confidence: 99%

Molecular Basis of Canine Muscle Type Phosphofructokinase Deficiency

Smith

Stedman

Rajpurohit

et al. 1996

Journal of Biological Chemistry

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Muscle type phosphofructokinase (M-PFK) deficiency is a rare inherited glycogen storage disease in humans that causes exertional myopathy and hemolysis. The molecular basis of canine M-PFK deficiency, the only naturally occurring animal homologue, was investigated. Lack of M-PFK enzyme activity was caused by a nonsense mutation in the penultimate exon of the M-PFK gene, leading to rapid degradation of a truncated (40 amino acids) and therefore unstable M-PFK protein. A polymerase chain reaction-based test was devised to identify M-PFK-deficient and carrier animals. This represents one of only a few inborn errors of metabolism where the molecular defect has been identified in a large animal model which can now be used to develop and assess novel therapeutic strategies.

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Genes transferred by retroviral vectors into normal and mutant myoblasts in primary cultures are expressed in myotubes.

Cited by 24 publications

References 5 publications

Adenovirus as an expression vector in muscle cells in vivo.

Adenovirus as an expression vector in muscle cells in vivo.

Expression of human factor IX in mice after injection of genetically modified myoblasts.

Molecular Basis of Canine Muscle Type Phosphofructokinase Deficiency

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