1061. Upstream Polyadenylation Enhancers in the U3 Region of Self_Inactivating Retroviral and Lentiviral Vectors Improve Titer and Gene Expression, and Prevent Transcriptional Read_Through
Authors: Axel Schambach, Melanie Galla, Baum Christopher
Journal: Molecular Therapy (2006)

Severe adverse events related to insertional mutagenesis have reinforced the interest in self_inactivating (SIN) retroviral vectors lacking enhancer_promoter sequences in the U3 region of the long terminal repeats (LTRs), thereby decreasing the likelihood of transactivation of neighboring alleles. However, deletion of the 3' U3 region may impede transcriptional termination, increasing the probability of read_through into cellular genes (Zaiss et al., J. Virol. 76, 2001). To improve 3' end processing, introducing strong heterologous polyadenylation signals within the R region or downstream of the 3′ LTR has been proposed. However, these modifications only improve polyadenylation in transfected packaging cells and are lost in transduced target cells. In contrast, including such sequences into U3 would act both during vector production and in transduced cells. We therefore incorporated 7 different upstream polyadenylation enhancer elements (USE) derived from viral (HIV, SV40, WHV, adenovirus) or cellular genes (thrombin, C2 complement) and cloned these into the 3′ U3 deletion of gammaretroviral SIN vectors. This resulted in an enhancement of both titer and gene expression in several cell lines and hematopoietic primary cells. Similar effects were observed in the context of 3 internal promoters (CMV, PGK, SFFV). A recombinant 100 bp sequence designed on the basis of the SV40 USE element gave the best results. Compared with the WHV posttranscriptional regulatory element (PRE), this element mediates approx. 50% of the enhancing activity on gene expression and titer. Importantly, the recombinant SV40 USE also prevents transcriptional read_through, in contrast to the PRE. This was confirmed by FACS and Northern blot assays, using a sensitive reporter assay in which a fluorescent protein is expressed from an internal ribosomal entry site inserted downstream of the 3′ LTR. Finally, the recombinant SV40 USE also enhanced titers and gene expression in 3rd generation lentiviral vectors lacking the PRE. Taken together, the presence of small USE elements in retroviral or lentiviral vectors can substitute for the presence of a PRE element, leading to a gain in titer and gene expression levels. The decreased likelihood of read_through over the termination signal potentially improves biosafety. Severe adverse events related to insertional mutagenesis have reinforced the interest in self_inactivating (SIN) retroviral vectors lacking enhancer_promoter sequences in the U3 region of the long terminal repeats (LTRs), thereby decreasing the likelihood of transactivation of neighboring alleles. However, deletion of the 3' U3 region may impede transcriptional termination, increasing the probability of read_through into cellular genes (Zaiss et al., J. Virol. 76, 2001). To improve 3' end processing, introducing strong heterologous polyadenylation signals within the R region or downstream of the 3′ LTR has been proposed. However, these modifications only improve polyadenylation in transfected packaging cells and are lost in transduced target cells. In contrast, including such sequences into U3 would act both during vector production and in transduced cells. We therefore incorporated 7 different upstream polyadenylation enhancer elements (USE) derived from viral (HIV, SV40, WHV, adenovirus) or cellular genes (thrombin, C2 complement) and cloned these into the 3′ U3 deletion of gammaretroviral SIN vectors. This resulted in an enhancement of both titer and gene expression in several cell lines and hematopoietic primary cells. Similar effects were observed in the context of 3 internal promoters (CMV, PGK, SFFV). A recombinant 100 bp sequence designed on the basis of the SV40 USE element gave the best results. Compared with the WHV posttranscriptional regulatory element (PRE), this element mediates approx. 50% of the enhancing activity on gene expression and titer. Importantly, the recombinant SV40 USE also prevents transcriptional read_through, in contrast to the PRE. This was confirmed by FACS and Northern blot assays, using a sensitive reporter assay in which a fluorescent protein is expressed from an internal ribosomal entry site inserted downstream of the 3′ LTR. Finally, the recombinant SV40 USE also enhanced titers and gene expression in 3rd generation lentiviral vectors lacking the PRE. Taken together, the presence of small USE elements in retroviral or lentiviral vectors can substitute for the presence of a PRE element, leading to a gain in titer and gene expression levels. The decreased likelihood of read_through over the termination signal potentially improves biosafety.