Gene Therapy Protocols; Volume 2: Design and Characterization of Gene Transfer Vectors

JAMES F CURTIN; MARIANELA CANDOLFI; MARIANA PUNTEL; WEIDONG XIONG; AKM GHULAM MUHAMMAD; KURT M KROEGER; CHUNYAN LIU; SONALI MONDKAR; NIYATI S BONDALE; PEDRO R LOWENSTEIN; MARIA G CASTRO

Methods Molecular Biology

HUMANA PRESS INC

Año: 2008; p. 239 - 266

  Regulatable promoter systems allow gene expression to be highly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases 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, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illnesses that require long-lasting gene expression. 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 control of the TeTON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes , immune stimulatory genes, and toxins for cancer gene therapeutics, i.e. glioma that ios a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (Dox)-sensitive rtTA2s-M2 inducer and tTSkid silencer, is non-toxic, relatively non-immunogenic and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and in vivo.