Large double copy vectors are functional but show a size-dependent decline in transduction efficiency.

The development of SIN-lentivectors has paved the way for the double copy vectors (DCV), which substitute the deletion in the 3'LTR with either transgenic or insulator sequences. However, the limits of this approach remain unclear. Previous results have demonstrated that transduction efficiencies of DCV carrying large insulator inserts in their 3'LTRs were impaired in a size-dependent manner. We wondered if this was also true for promoter-transgene inserts and whether they remained functional upon integration into the genome. Therefore, we designed a series of DCV with increasing 3'LTR sizes containing different promoter-transgene combinations. Transduced cells were scored for both transduction efficiency and insert functionality. We found that the transduction efficiencies indeed were impaired in a size-dependent way. Efficiency with inserts below 1 kb linearly decreased with size, while sizes between 1 and 2 kb showed a further decrease to a minimum of 5% of an original "empty" SIN-vector. However, we did not find an LTR size that completely abolished transduction. Moreover, we demonstrated that all inserts remained functional regardless the promoter-transgene combination used. Therefore, we conclude from our data that DCV indeed remain functional, but transduction efficiencies drop radically when inserts larger than 1 kb are being used.