A System for Small-Molecule Control of Conditionally Replication-Competent Adenoviral Vectors

Abstract

Replication-competent adenoviral vectors are potentially far more efficient than replication-defective vectors. However, for reasons of safety, there is a need to restrict viral replication both spatially, by limiting replication to certain cell types, and temporally. To control replication temporally, we have developed a system, based on the small-molecule dimerizer rapamycin, for regulating the replication of adenoviral vectors. In this system, one adenoviral vector, AdC4, expresses transcription factors whose activity is regulated by the non-immunosuppressive rapamycin analog AP21967. A second vector, Ad(Z12-I-E1aE1b19k), contains E1 genes placed downstream of binding sites for the regulated transcription factor. Co-infection of several cell lines by the vector pair leads to dimerizer-dependent E1 expression and an increase in viral replication, as shown by Southern blots and replication assays. Furthermore, expression of a reporter gene from a replication-defective vector, Ad-GM-CSF, can be augmented by up to 18-fold by co-infection with the pair of conditionally replicating vectors in the presence of dimerizer. Similar results are obtained when the vectors are directly injected into subcutaneous HT1080 xenograft tumors in nude mice. We believe that vectors based on this principle will be a useful additional tool to enhance efficiency and safety of gene delivery for anti-cancer therapy.

Tamoxifen-regulated adenoviral E1A chimeras for the control of tumor selective oncolytic adenovirus replication in vitro and in vivo

“…All of them are based on the regulation of E1A gene expression, either through the use of promoters directly Tamoxifen-dependent control of oncolytic adenovirus replication I Sipo et al dependent on certain drugs for their activity 20 or the use of different combinations of drug-dependent transcriptional transactivator (TA) and/or silencer (TS) proteins with their corresponding responsive elements. [21][22][23] In their present form, 10,24 these oAdVs have several disadvantages. An MMTV promoter-dependent oAdV, for example, requires dexamethasone for drug-dependent activation of E1A expression.…”

“…20 Dexamethasone affects the metabolism of many organs, which makes it difficult to use it in clinical applications. Recently, we have used the Tet-On system 22,23 and other investigators have turned to the rapamycin-dependent dimerizer gene expression system 21 for control of oAdV replication. Presently, however, these oAdVs require one or two additional vectors expressing the drug-dependent transcriptional regulators.…”

“…Furthermore, compared with gene expression systems previously examined for control of oAdVs. [21][22][23] E1A-Mer chimeras simplify the construction of pharmacologically regulated oAdVs. The two E1A-Mer variants MEM and E DNLS M best fulfil the requirements of tightly regulated effector proteins for adenoviral replication and oncolytic activity.…”

“…One promising approach appears to be the use of oAdVs, whose replication can be regulated pharmacologically, opening the possibility to control adenoviral replication temporally from the outside. To this end, it has been shown that adenoviral replication can be pharmacologically regulated via control of E1A gene expression by a mouse mammary tumor virus (MMTV) promoter, 20 by the rapamycin dimerization system, 21 by an optimized Tet-On gene expression system 22,23 or through insertion of drug responsive elements into tumor-specific promoters. 10,24 An alternative strategy to control gene function is based on direct regulation of the activity of an effector protein, by fusing it to the hormone-binding domain (HBD) of steroid receptors.…”

Heat-directed suicide gene therapy for breast cancer

“…the prostatespecific antigen gene promoter 39 ) or ligand-inducible (e.g. rapamycin 40 ) promoter systems to drive the expression of E1 genes. These vectors would be expected to improve therapeutic outcome significantly since adenovirus replication is in itself oncolytic and would result in amplification of the therapeutic gene and further dissemination of the virus within the tumor.…”