The universal lentiviral packaging cell

Inherited diseases are caused by mistakes in the DNA of patients. In the case of rare inherited immunodeficiencies diseases (Baby in the Bubble syndrome), they can be cured by delivering a correct copy of the faulty DNA using an engineered virus (gene therapy). This project is to be carried out by scientists in the MRC/UCL Medical Molecular Virology Centre at UCL. They will develop a safer and more efficient method to produce engineered viruses for clinical gene therapy trials.

Lentiviral vectors integrate and express transgenes in most cells. Due to cytotoxicity of viral proteins vectors are currently produced by transient transfection. This is costly, laborious, inherently unsafe and hard to scale-up. We recently reported a prototype packaging cell that continuously produced high-titre lentiviral vectors (see key reference). We now request funding to produce a safe, easily manipulated packaging system suitable for clinical lentiviral vector production by the following strategy, using recombination mediated cassette exchange:
1. Infect 293T cells with an MLV vector containing a loxP site in LTR, then transient transfection of Cre recombinase and selectable lentiviral Gag-Pol cassettes.
2. Infect these cells with a second MLV vector containing an FRT site, then transfected with Flp recombinase and selectable lentiviral vector cassettes.
3. Gammaretroviral envelopes will be expressed, viral safety and immunogenicity will be tested.