Transcriptional Enhancers Induce Insertional Gene Deregulation Independently From the Vector Type and Design

Abstract

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…Extensive studies have shown that the enhancer sequences are the major cause of cell transformation, making the design of viral vectors for life-long therapy approaches even more challenging. [8][9][10] Insulators mark the boundaries of chromatin domains and limit the range of action of enhancers and silencers. 11 They are characterized by at least one of the following properties: Enhancer blocking and/or boundary.…”

Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts

“…The strong transcriptional enhancer present in the MLV long terminal repeat (LTR) played a major role in deregulating gene expression. Preclinical studies showed that enhancer-less (self-inactivating [SIN]), HIV-derived LVs are less likely to cause insertional tumors than MLV-derived vectors (17)(18)(19)(20). Transcriptional gene activation, however, is not the only genotoxic event that may result from retroviral vector integration.…”

Retroviral integration profiles: their determinants and implications for gene therapy

“…In contrast, HIV-1 disfavors CpG islands and does not show any preference for TFBSs (16,22,26). Interestingly, integrated lentiviral vectors can both up-and down-regulate host gene expression, in contrast to MLV (20). Another lentivirus, simian immunodeficiency virus (SIV), also shows a similar integration pattern of favoring actively transcribed genes with no preference for TSSs and CpG islands (19,28,29).…”

“…Activated oncogenes often turn normal cells into can-cerous cells by perturbing the regulation of cell growth and survival. The retroviral promoter and enhancer components have recently been shown to affect cellular transcription over a long range, independent of vector type, thereby increasing the tumorigenicity of the viral vector (20). The retroviral propensity for frequently targeting oncogenes and cell growth/survival genes and the integration preference for TSSs and CpG islands synergistically increase the malignant potential of retroviral vectors (21).…”