Two-Step Delivery of Retroviruses to Postmitotic, Terminally Differentiated Cells

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Abstract
Recombinant replication-defective retroviral vectors are currently the most commonly used vectors for introducing foreign genes into human cells in gene therapy protocols. Their genomes stably incorporate in the host chromosomes of mitotic cells, thus ensuring stable expression. However, the applications of retroviruses to gene therapy are limited by their inability to infect postmitotic cells such as muscle fibers. In an attempt to overcome such limitations, we have developed a novel two-step transduction protocol that allows integration and expression of retroviral genes in differentiated cells. We induced DNA synthesis in terminally differentiated cultured mouse myotubes derived from both established myogenic cell lines and from primary myoblasts. We infected the postmitotic cells with a recombinant replication-defective adenoviral vector encoding the SV40 large T antigen as a mitogen. Subsequently we transduced the adenovirus-infected cells with a Moloney retroviral vector bearing the LacZ gene. Histochemical analysis revealed the coincident expression of LacZ gene in those myotubes that had been induced to synthesize DNA. Retroviruses are the most commonly used viral vectors in gene therapy to transfer foreign genes into living cells. However, they have some major disadvantages including the limitation that they can only infect dividing cells. Ito and Kedes demonstrate the use of a cell cycle inducer, SV40 large T antigen, to induce DNA synthesis in postmitotic, terminally differentiated skeletal muscle cells. This first step renders the cells permissive for subsequent retroviral transduction and expression.