Enhanced transcriptional insulation of lentiviral vectors using "sequence-upgraded polyA long terminal repeats" or...

The safety and quality attributes of viral vector-based products are of paramount importance. Recent successful commercialized use of lentiviral vectors (LVs) ex vivo anticipates future, higher dose applications in vivo, leading to greater integration events per patient. Contemporary LVs employ "self-inactivating" (SIN) long terminal repeats (LTRs) that are deleted for enhancer-promoter sequences, which greatly minimizes the probability of insertional oncogenesis. However, such deletion also removes polyadenylation enhancer sequences, leading to reduced polyadenylation efficiency. Transcriptional read-in/out of integrated SIN-LVs typically results in interaction of LV sequences with the cellular transcriptome. Here we developed "sequence-upgraded polyA-LTRs" (supA-LTRs) that have ∼70-fold and ∼100-fold increased inherent polyadenylation activity in sense and antisense directions, respectively. This was achieved by redefinition of the polyA signal position with respect to the R region and optimal spacing of heterologous upstream and downstream enhancers, completely decoupling transcriptional termination from native HIV-1 polyadenylation sequences. The greatest block (>20-fold) to 5' transcription read-in to the integrated LV cassette was observed when the supA-LTR was combined with LVs that have an inactivated major splice donor site ("SupA2KO-LVs"). SupA-LTRs also mediated an ∼2-fold increase in transgene expression in primary cells. SupA-LTRs represent a further step forward providing additional safety and utility to the next generation of LVs.