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Content archived on 2024-05-07

Genetically modified skeletal muscle cells as a tool gene replacement in genetic disorders.

Objective



Skeletal muscle is an important target for human somatic cell gene therapy applications. These include systemic delivery of recombinant gene products for correction of genetic or acquired disorders, as well as gene therapy of inherited muscle diseases. The aim of this project is the development of safe, efficient and reliable methodologies for gene transfer into human muscle cell progenitors, to obtain stable and regulated production of recombinant proteins into, or by, skeletal muscle fibres. Vectors, gene transfer technology and pre- clinical models will be developed to test the use of genetically modified fibres to produce either circulating proteins of biological interest (e.g. hormones, enzymes, cytokines), or structural or enzymatic proteins missing or abnormal in congenital muscle disorders (e.g. dystrophin). The experimental strategies will be specifically aimed to obtain regulated expression of the transferred gene for systemic protein delivery, and on production of significant numbers of genetically modified muscle fibres for therapy of primary myopathies. Most of the work will be carried out on primary human muscle cell progenitors, which will be transduced in-vitro mainly, although not exclusively, by replication- defective retroviral vectors. Transduced cells will be studied both in cell culture and in-vivo, after transplantation into regenerating muscle of immunodeficient mice to generate human-mouse hybrid fibres. For systemic delivery of circulating proteins (al-antitrypsin and IL-4 as initial models), we will study stability, long-term persistence and quantitative expression of genes transduced in a variety of retroviral vectors. Development of muscle-specific vectors producing the necessary amount of protein under the regulation required for each specific application is a major target of this project. The combined expertise of the applicants in muscle-specific gene regulation, and in retroviral vector construction and utilization, will be instrumental and crucial to the success of the proposed research. For reconstitution of muscle tissue in primary myopathies with genetically modified fibres, we will attempt to scale-up the number of transduced re-implantable myogenic cells by a variety of experimental strategies, including conditional or reversible immortalization by SV40 T-antigen, and recruitment of mesodermal cells (thymus, skin, bone marrow, etc.) which can be converted to myogenesis. Although the general feasibility of the gene therapy approach to muscular dystrophies has been demonstrated, availability, delivery, survival, and colonization ability of large numbers of transduced myogenic cells are all crucial issues which need be addressed in practical terms. We will test a number of alternative experimental approaches thoroughly and in a quantitative fashion, using the human-mouse hybrid as an in- vivo, pre-clinical model. In the specific case of Duchenne Muscular Dystrophy, the efficiency of mini-dystrophin will be compared with that of alternative proteins. The applicant groups have pioneered the field of gene and cell therapy of muscular dystrophy, and are in the best position to provide the cumulative effort required for completion of a research effort that could not be accomplished by a single laboratory working on a national scale. This research effort is expected to produce a large amount of information which will provide the basis for the planning and initiation of clinical trials for a variety of different human pathologies.

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Coordinator

Università degli Studi di Roma "La Sapienza"
EU contribution
No data
Address
14,Via Scarpa
00161 Roma
Italy

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Total cost

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Participants (6)

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