Comparative Genomic Integration Profiling of Sleeping Beauty Transposons Mobilized With High Efficacy From Integrase-defective Lentiviral Vectors in Primary Human Cells

Abstract

It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer.

In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors

“…These CFU colonies were obtained from Lin 2 /GFP 1 BM cells of in vivo transduced hCD46tg animals. Amplification of SB genomic DNA junctions was performed by linear amplification-mediated polymerase chain reaction (PCR) as described previously 14 and in detail in the supplemental Methods (see "Construction of integration site libraries").…”

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…When performing transgenesis, a single copy transgene insertion, which is not disturbing endogenous gene functions is desirable. Chromosomal integration of SB transposons is nearly random at the genome level resulting in ~60 % of the SB transposon integrations being intergenic [32][33][34][35] . Because the transposase is only transiently present in the cell, the integrated transposable element is stable (i.e., will not undergo further rounds of transposition), thereby rendering transposons easily controllable, conditional DNA delivery tools that can be used for versatile applications, including germline gene transfer.…”

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…In contrast to the considerable specificity at the primary DNA sequence level, SB integration can be considered fairly random at the genomic level (Vigdal et al, 2002;Yant et al, 2005;Moldt et al, 2011). Roughly one-third of SB insertions in mouse and human cells occur in transcribed regions, and because genes cover about one-third of the genome, such frequency suggests neither preference for nor disfavoring insertion into genes.…”

“…Alternatively, the development of hybrid vector systems combining the natural ability of viruses to traverse cell membranes with efficient genomic insertion mediated by the SB system is a promising strategy. Indeed, components of the SB transposon have been incorporated into integrase-defective lentiviral particles that showed efficient gene transfer in a range of human cell types and an insertion profile favorable to conventional lentiviral vectors (Staunstrup et al, 2009;Vink et al, 2009;Moldt et al, 2011). Hybrid adenovirus-SB vectors (Yant et al, 2002) have been used to efficiently deliver SB transposon vectors expressing FIX into the liver in a hemophilic dog model (Hausl et al, 2010).…”