Lentivector-mediated transfer of Bmi-1 and telomerase in muscle satellite cells yields a duchenne myoblast cell line with long-term genotypic and phenotypic stability.

Conditionally immortalized human cells are valuable substrates for basic biologic studies, as well as for the production of specific proteins and for the creation of bioartificial organs. We previously demonstrated that the lentivector-mediated transduction of immortalizing genes into human primary cells is an efficient method for obtaining such cell lines. Here, we used human muscle satellite cells as model targets to examine the impact of the transduced genes on the genotypic and phenotypic characteristics of the immortalized cells. The most commonly used immortalizing gene, the SV40 large T antigen (T-Ag), was extremely efficient at inducing the continuous growth of primary myoblasts, but the resulting cells rapidly accumulated major chromosomal aberrations and exhibited profound phenotypic changes. In contrast, the constitutive expression of telomerase and Bmi-1 in satellite cells from a control individual and from a patient suffering from Duchenne's muscular dystrophy yielded cell lines that remained diploid and conserved their growth factor dependence for proliferation. However, despite the absence of detectable cytogenetic abnormalities, clones derived from satellite cells of a control individual exhibited a differentiation block in vitro. In contrast, a Duchenne-derived cell line exhibited all the phenotypic characteristics of its primary parent, including an ability to differentiate fully into myotubes when placed in proper culture conditions. This cell line should constitute a useful reagent for a wide range of studies aimed at this disease.

Transduction of myogenic cells by retargeted dual high-capacity hybrid viral vectors: robust dystrophin synthesis in duchenne muscular dystrophy muscle cells.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), making it amenable to gene- or cell-based therapies. Another possible treatment entails the combination of both principles by transplantation of autologous myogenic cells after their genetic complementation. This approach requires efficient and stable transduction of these cells with recombinant DMD. Recently, we generated a dual high-capacity (hc) adenovirus (Ad)-adeno-associated virus (AAV) hybrid vector (HV) that can deliver two full-length dystrophin-encoding modules into target cells. We showed that HV transduction of human cells containing AAV Rep proteins leads to the insertion of foreign DNA into the AAVS1 locus. Here, we improved HV entry into muscle cells from DMD patients. After having verified that these cells barely express the coxsackie B virus and Ad receptor (CAR), which constitutes the attachment molecule for Ad serotype 5 (Ad5) fibers, we equipped dual hcAd/AAV HV particles with Ad serotype 50 fiber domains to achieve CAR-independent uptake. These retargeted vectors complemented much more efficiently the genetic defect of dystrophin-defective myoblasts and myotubes than their isogenic counterparts with conventional Ad5 fibers. Importantly, the accumulation of beta-dystroglycan along the membranes of vector-treated DMD myotubes indicated proper assembly of dystrophin-associated glycoprotein complexes.

A role for atm in E-cadherin-mediated contact inhibition in epithelial cells.

Ataxia telangiectasia is a hereditary pleiomorphic syndrome caused by loss of Atm, a phosphoprotein involved in multiple signaling pathways. Here, we propose a novel role for atm in cultured epithelial cells, namely the regulation of cell growth by contact inhibition. We show that atm is upregulated in epithelial cells reaching confluence. Conditional expression of the PI 3-Kinase domain of atm in non-confluent Tac-2 epithelial cells increases the expression of the anti-proliferative gene Tis-21 and downregulates key cell cycle regulator genes, such as cyclins A, B1, B2, E and E2. Finally, we demonstrate that upregulation of atm, and thus Tis-21, in confluent Tac-2 cells can be inhibited by an E-cadherin antibody blocking specifically homophilic E-cadherin interactions between adjacent cell surfaces. Altogether, these results suggest that atm could participate in a molecular pathway linking extracellular signalling to cell cycle control and may help further clarify the role of Atm in epithelial cell biology and carcinogenesis.