Regulated expression of adenoviral vectors-based gene therapies: therapeutic expression of toxins and immune-modulators

CURTIN; CANDOLFI; PUNTEL; XIONG; MUHAMMAD; KROEGER; MONDKAR; LIU; BONDALE; LOWENSTEIN; CASTRO

METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.)

CLIFTON, N.J.

Año: 2008 p. 239 - 266

1064-3745

SummaryRegulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highlydesirable for the development of safe, efficacious adenoviral vectors that can be used to treat humandiseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF”state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, thecomponents of regulatable cassettes should be non-toxic at physiological concentrations and shouldnot be immunogenic, especially when treating chronic illness that requires long-lasting geneexpression. In this chapter, we will describe in detail protocols to develop and validate first generation(Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the controlof the TetON regulatable system. Our laboratory has successfully used these protocols to regulatethe expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics,i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetONregulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKidsilencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgeneexpression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo.