Efficient SB100X-directed transposition from non-integrating lentiviral vectors in primary human cells

Biological properties of the integration machinery of lentiviral vectors facilitate preferred insertion into actively transcribed genomic regions. We have previously shown that DNA circles generated during lentiviral transduction can serve as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) DNA transposase. Notably, insertion by this engineered pathway of integration was random and not biased toward genes. Here, we describe the enhanced gene transfer properties of a new combined HIV-1/SB vector system that allows efficient DNA transposition from non-integrating lentiviral vectors in a variety of cell types. Transposition directed by the hyperactive SB100X transposase is further enhanced by including improved SB T2 inverted repeats in the vector. With this vector system, we achieve a transduction rate that is increased up to 80-fold relative to background in human embryonic kidney cells (HEK293) and more than 30-fold in myelogenous leukemia cells (K562). Moreover, highly efficient gene insertion in both primary human fibroblasts and primary human keratinocytes, up to 160-fold above the background level, is reported. In summary, the combined HIV-1/SB vector system integrates genetic cargo with high efficacy, in some cases with almost the same efficiency as conventional lentiviral vectors. Our data suggest that the biological constraints on conventional lentiviral DNA insertion are resolved by the SB100X transposase, resulting in a random integration profile. We believe that such vectors may become valuable tools for genetic engineering and therapeutic gene transfer.