Sleeping Beauty Transposition From Nonintegrating Lentivirus

Abstract

Lentiviral vectors enter cells with high efficiency and deliver stable transduction through integration into host chromosomes, but their preference for integration within actively transcribing genes means that insertional mutagenesis following disruption of host proto-oncogenes is a recognized concern. We have addressed this problem by combining the efficient cell and nuclear entry properties of HIV-1-derived lentiviral vectors with the integration profile benefits of Sleeping Beauty (SB) transposase. Importantly, this integration enzyme does not exhibit a preference for integration within active genes. We generated integrase-deficient lentiviral vectors (IDLVs) to carry SB transposon and transposase expression cassettes. IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats (IRs) but devoid of HIV-1 long terminal repeats (LTRs) or other virus-derived sequences. Importantly, integration site analysis revealed redirection toward a profile mimicking SB-plasmid integration and away from integration within transcriptionally active genes favored by integrase-proficient lentiviral vectors (ILVs).

Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy

“…Unlike gammaretrovirus and lentivirus that have the propensity to integrate the transgenes into the transcription start sites or transcription units, 42 the SB transposon results in fairly unbiased genomic integration, which reduces the risk of insertional mutagenesis and has been coupled with the lentiviral vector to redirect the vector integration preference. 43 Moreover, SB seems to trigger significantly milder epigenetic changes at the genomic insertion site, hence minimizing the potential genotoxicity. 36 The SB-baculovirus vector took advantage of the efficient baculovirus transduction, alleviated the shortcoming of conventional baculovirus vectors (that is, transient expression) and augmented the antitumor effects by expressing the antiangiogenic hEA protein.…”

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…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).…”

Integrase-defective lentiviral vectors: progress and applications

“…An alteration in any of these residues inactivates the catalytic properties of IN. Of these potential mutation target sites, D64 is most commonly altered, 13,22,44,[48][49][50][51][52][53][55][56][57]60 but mutations to D116 have also been reported. 51,54,58,59 Additional mutations affect IN DNA binding, linear episome processing or IN multimerization ( Figure 2).…”

“…53 Two recent reports described Sleeping Beauty (SB) transposase-mediated transposition when delivered using an IDLV. 57,60 Unlike retroviral integration, SB transposition shows little preference for specific genomic regions. 57,60 Both studies constructed two IDLVs, one expressing a hyperactive SB transposase and a second containing a transposable element with a drug resistance transgene cassette and necessary inverted repeats.…”

“…[47][48][49] Several reports have detailed the production of HIV IDLVs. 13,22,44,[47][48][49][50][51][52][53][54][55][56][57][58][59][60] In addition, feline immunodeficiency virus (FIV) IDLVs are also described. 48 Integrase-defective LVs: progress and applications MB Banasik and PB McCray Jr studies ( Figure 1).…”