A Rapid and Quantitative Assay for Measuring Neighboring Gene Activation by Vector Proviruses

Abstract

A simple, quantitative assay for measuring the oncogenic potential of integrating vectors is needed in order to improve vector design and safety. In this study, we have developed a transient plasmid-based assay to measure the activation of a reporter gene by an adjacent vector provirus. Plasmid pACT contains a luciferase cassette driven by a minimal, enhancerless promoter, into which vector proviruses are inserted upstream for evaluation by luciferase assays and northern blots. In a comparison of analogous vectors based on murine leukemia virus (MLV), human immunodeficiency virus (HIV), and foamy virus (FV), we observed significant enhancer activity and read-through transcription from MLV proviruses, and significant read-through transcription from HIV proviruses. HIV and FV proviruses containing an internal MLV long-terminal repeat (LTR) promoter also had significant enhancer activity, which was not observed with an internal promoter from the murine phosphoglycerate kinase-1 gene, PGK. These results demonstrate that neighboring gene activation can be limited by using internal promoter(s) lacking enhancer activity, especially when present in an FV vector backbone that prevents read-through transcription. Although the pACT assay does not measure oncogenesis directly, it should be useful for screening vectors before more time-consuming and costly animal studies are undertaken.

Foamy virus vectors: The usefulness of a perfect parasite

“…Moreover, silencing of transgenes expressed from FV vectors has not been observed yet, indicating that FV vectors are suited for applications where long-term expression is a desired feature. 1, [32][33][34] Recently, in another study addressing safety, Hendrie et al 35 developed a plasmid-based assay to estimate the likelihood of an integrated retroviral vector to stimulate transcription of a nearby (indicator) gene with a minimal promoter, either by enhancer effects of transcriptional control elements in the vector backbone or by read-through from the long terminal repeat or internal promoter. Both phenomena are implicated in the activation of cellular proto-oncogenes by retroviral vectors.…”

Insulated Foamy Viral Vectors

“…20,21 In addition to a more desirable integration profile, FV vectors also have a lower propensity to dysregulate nearby genes when directly compared with GV and LV vectors. 25 These increased safety features, in addition to a large therapeutic transgene-carrying capacity 17 and broad tissue tropism including human mobilized peripheral blood and cord blood-derived CD34 + cells, 23,26 suggest that FV vectors are a promising vector for safer HSC gene therapy.…”

Risk assessment in skin gene therapy: viral–cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors

“…g-Retroviral (Moloney leukemia virus)-derived vectors displayed high frequency of 3¢ polyadenylation signal readthrough, as shown in transient transfection assays of retroviral vector constructs carrying a reporter expression cassette situated downstream from the vector sequence. 10,11 U3 deletion in SIN lentiviral vectors results in decreased RNA 3¢ processing and the increased generation of chimeric transcripts, 12 demonstrating that the U3 enhancer deletion, aimed at mitigating insertional mutagenesis by enhancer transactivation, might be the source of potentially troubling unintended transcription of viral-cellular fusion genes.…”