Improving Transcriptional Termination of Self-inactivating Gamma-retroviral and Lentiviral Vectors

Abstract

Adverse events relating to insertional mutagenesis have reinforced the interest in self-inactivating (SIN) gamma-retroviral and lentiviral vectors without enhancer-promoter sequences in the U3 region of the long terminal repeats. However, SIN vectors suffer from leaky transcriptional termination, increasing the probability of read-through into cellular genes. To improve 3' end processing, we incorporated seven upstream polyadenylation enhancer elements (or upstream sequence elements, USEs) derived from viral or cellular genes into the 3' U3 region of gamma-retroviral and lentiviral SIN vectors. A 100-base-pair sequence representing a recombinant direct repeat of the USE derived from simian virus 40 (2xSV USE) gave the best results, improving both titer and gene expression. In both gamma-retroviral and lentiviral SIN vectors, the 2xSV USE partially substituted for effects provided by the much larger post-transcriptional regulatory element derived from woodchuck hepatitis virus (wPRE). By northern blot and reporter assays, we found that the 2xSV USE greatly improved proper messenger RNA (mRNA) processing at the retroviral termination signal. Importantly, the 2xSV USE was superior to the wPRE in suppressing transcriptional read-through, improving not only vector efficiency but potentially also biosafety.

Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS

“…The titers of these various vector batches were comparable and were not significantly different if the WPRE was present or not (Table 2). These results are consistent with those of Schambach et al 8 reporting that variable Validation of a mutated PRE MA Zanta-Boussif et al effects of the WPRE on titers were observed according to the LV construct. The effects of the WPRE on transgene expression in target cells were examined following transduction of WAS patient B-cell lines with the various vectors containing or not containing WPRE and by measuring protein levels in western blots (Figures 4a and b).…”

“…The WPRE seems to increase the amount of polyadenylated transcripts and clearly augments the size of the polyA tail of RNA. 8 The polyA motifs are contained in the long terminal repeats (LTRs) of gammaretroviral or of human immunodeficiency virus (HIV)-1-derived gene transfer cassettes. When placed upstream of the 3 0 LTR, the WPRE improves transcript termination and therefore can significantly reduce transcript read-through, particularly with Moloney-derived vectors.…”

“…When placed upstream of the 3 0 LTR, the WPRE improves transcript termination and therefore can significantly reduce transcript read-through, particularly with Moloney-derived vectors. 6,8 Probably in part through this effect, the WPRE can also augment the titers of viral preparations although results are variable and the exact mechanisms underlying this effect remain to be determined. 6,8 The magnitude of the effects of WPRE vary upon the specific context of the promoter, transgene and cell type, and this has been shown both for its effects on transgene expression 4 or viral titers.…”

“…6,8 Probably in part through this effect, the WPRE can also augment the titers of viral preparations although results are variable and the exact mechanisms underlying this effect remain to be determined. 6,8 The magnitude of the effects of WPRE vary upon the specific context of the promoter, transgene and cell type, and this has been shown both for its effects on transgene expression 4 or viral titers. 3,6,8 For this reason, the effects of this 3 0 regulatory element need to be evaluated in specific contexts.…”

“…6,8 The magnitude of the effects of WPRE vary upon the specific context of the promoter, transgene and cell type, and this has been shown both for its effects on transgene expression 4 or viral titers. 3,6,8 For this reason, the effects of this 3 0 regulatory element need to be evaluated in specific contexts.…”