Enhanced transcriptional insulation of lentiviral vectors using “sequence-upgraded polyA long terminal repeats” or “supA-LTRs”

, 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.