Insertion of the 1.2kb Chicken β-Globin 5′HS4 Insulator Element in the LTR Inhibits HIV-1 Vector Mediated Transduction at the Early Infection Step.

Insertional oncogenesis is the major concern in gene therapy using integrating vectors such as gamma-retrovirus vectors and lentivirus vectors. One promising strategy to circumvent this unwanted event is the use of insulator elements which can block trans-activation of proto-oncogenes by enhancers of the integrated vectors. The 1.2kb fragment containing the chicken β-globin 5′ hypersensitivity site 4 (5′HS4) is the most well characterized insulator element. The insulator elements are usually inserted into the U3 region of the 3′ LTR of the vector construct, because the insulator element is duplicated and transferred to the 5′ LTR during viral replication. In the transduced cells, two insulator elements are located outside the retroviral transcriptional unit and this configuration is thought to be essential for efficient enhancer blocking. However, we have previously shown that insertion of the 1.2kb 5′HS4 element in the LTR of SIN-HIV-1 vector significantly reduced the transduction efficiency assayed by GFP expression in HeLa cells. In the present experiments, we examined the mechanism of reduced biological titer. We compared SIN-HIV-1 vectors with (INS) or without (PARENT) the 1.2 kb insulator element. The biological titer on HeLa cells (TU/ml), p24Ag concentration (ng^p24/ml), and virus RNA genome content (vg copy/ng^p24) of these vectors were shown in Table 1. As we previously reported, the biological titer of INS was significantly lower than that of PARENT, although p24 and RNA contents were not influenced by insertion of the 1.2 kb insulator element. In the next experiment, we performed reverse-transcription kinetic assay. Genomic DNA and low molecular weight DNA extracted by Hirt’s method were prepared from transduced HeLa cells and the amounts of reverse-transcribed vector sequence were determined by qPCR. The data showed that reverse-transcription of INS was remarkably reduced compared to PARENT. These results suggest that insertion of the 1.2 kb insulator does not inhibit production of virus particles and packaging of RNA genome. The responsible step for reduced titer of INS appears to be somewhere from the cellular entry step to the reverse-transcription step.

Characterization of HIV-1 vector with the 1.2 kb 5′HS4 insulator