239. Human Myoblast Deimmortalization Using Tat-Mediated Cre Recombinase Delivery

The reduced proliferating capacity of myoblasts isolated from Duchenne muscular dystrophy (DMD) patients limits our capacity to genetically modify and proliferate them in vitro for their use in autologous transplantation. Previously, our research group has successfully immortalized and extensively proliferated DMD myoblasts using the SV-40 Large T antigen (TAg) and hTERT1,2. Using a retroviral vector coding for TAg flanked by LoxP sites, immortalization reversal can be performed by Cre delivery. To circumvent the requirement for an additional infection, we used a Tat-Cre recombinase fusion protein to exert the recombination necessary for immortalization reversal. Tat-Cre intracellular transduction produced site-specific recombination and excision of the TAg. To facilitate the TAg excision process, cell lines containing a single immortalizing integrative event were generated. Using these cell lines, we demonstrate that by optimizing the delivery using a high concentration Tat-Cre protein, by co-incubating with chloroquine and by selecting against cells containing copies of the unrecombined vector, complete TAg removal could be achieved with a single 1 h treatment. In addition to the molecular evidence demonstrating immortalizing cassette removal, TAg excision also resulted in growth arrest within 2 days. The resulting cell culture could be maintained for at least 2 weeks. These results indicate that the intracellular delivery of a recombinant Tat-Cre protein could be a useful tool for a variety of applications that necessitate the manipulation of cells in culture.