DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors

Abstract

DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.

Delivering the Goods for Genome Engineering and Editing

“…This may for example result in variable copy numbers of inserted transposon vectors. 47 Heavily transfected cells are likely to undergo the intended genetic modification, but may also sustain the production of the genome tool for days. In our hands, activity of piggyBac transposase encoded by plasmid transfected into human cells lines can be detected up to 14 days This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.…”

“…110 Our own endeavors to develop lentiviral protein transduction for genome engineering were directed first at incorporating the hyperactive piggyBac transposase hyPBase 113 in lentiviral particles. 47 A schematic representation of this approach is provided in Figure 2. Using the packaging construct design developed by Aoki et al, 103 we fused hyPBase to the N-terminal end of Gag and separated the two domains with a HIV-1 protease cleavage motif.…”

“…81 We now know that the piggyBac transposase may equally well utilize IDLV-delivered substrates for DNA transposition. 47 Along the same lines, episomal DNA intermediates established by reverse transcription of IDLV-delivered vector RNA serve as effective donors for HDR by homologous recombination. 63,69 It has been suggested, though, that the use of free-ended nonviral or IDLV DNA as donors for homologous recombination may be associated with additional illegitimate recombination events.…”

Genetic Modification of T Cells

“…One possible way to overcome this limitation is to use non-integrating lentiviral vectors to deliver both the transposon and the transposase. This type of system has been successfully implemented in primary human cells to drive transposition using the PB system [53]. Of note, when this system was compared to electroporation delivery, it was demonstrated that transfected cell populations contained cells with multiple integration events, while virally transduced populations contained cells with only a single integration event.…”

“…Of note, when this system was compared to electroporation delivery, it was demonstrated that transfected cell populations contained cells with multiple integration events, while virally transduced populations contained cells with only a single integration event. This was likely due to the much lower level of transposase expression achieved with the viral delivery system [53,54]. While the efficiency of this system is still significantly lower than gene delivery using an integrating viral vector, the ability to limit integration events to one per cell will reduce the potential for genotoxicity in target cells.…”