Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

Abstract

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdDeltaE1AIL), the early gene E2B (AdDeltaE2BIL), or the late fiber gene (AdDeltafiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdDeltaE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdDeltaE1AIL) to six (AdDeltafiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdDeltaE2BIL, AdDeltafiberIL, and AdTL. AdDeltafiberIL and the parental virus, Ad5-Delta24, resulted in similar cytotoxicity, but AdDeltaE2BIL and AdDeltaE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdDeltaE2BIL and AdDeltafiberIL, but substantial for AdDeltaE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression.

Activation of the JAK-STAT3 pathway is associated with the growth of colorectal carcinoma cells

“…Simultaneously, the SOCS3 gene was fused with the cpp gene (cell-penetrating peptide), which has the capacity to deliver peptides, proteins, and oligonucleotides into intact cells (22,23). In addition, in the backbone of the adenovector, expression of the SOCS gene was controlled by an endogenous adenoviral major late promoter (MLP) which restricts SOCS3 expression within the tumor microenvironment due to tumor-specific viral replication (24). A previous study showed that this recombination manipulation of the oncolytic vector did not affect replication of the virus (10).…”

Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells

“…Quantification of mRNA copy numbers was performed by real-time PCR as previously described. 34 For the assay, known amount of template DNA of pTG3602 (10 8 , 10…”

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

“…IRESs are highly structured viral sequences that facilitate bicistronic gene expression by internal translation initiation in addition to translation initiation at the 5' end of mRNAs. For example, the approximately 600-bp encephalomyocarditis virus (EMCV) IRES has been used for transgene insertion into both early and late Ad transcription units (Sauthoff et al, 2002;Rivera et al, 2004). Alternative splicing is widely utilized by Ads as a mechanism for expression of multiple proteins from one transcription unit.…”