Targeted delivery of in situ PCR-amplified Sleeping Beauty transposon genes to cancer cells with lipid-based nanoparticle-like protocells

Abstract

A Sleeping Beauty (SB) transposon system is made of a transposon plasmid (containing gene encoding a desired functional or therapeutic protein) and a transposase plasmid (encoding an enzyme capable of cutting and pasting the gene into the host cell genome). It is a kind of natural, nonviral gene delivery vehicle, which can achieve efficient genomic insertion, providing long-term transgenic expression. However, before the SB transposon system could play a role in promoting gene expression, it has to be delivered efficiently first across cell membrane and then into cell nuclei. Towards this end, we used a nanoparticle-like lipid-based protocell, a closed bilayer of the neutral lipids with the DNA encapsulated inside, to deliver the SB transposon system to cancer cells. The SB transposon system was amplified in situ inside the protocells by a polymerase chain reaction (PCR) process, realizing more efficient loading and delivery of the target gene. To reach a high transfection efficiency, we introduced two targeting moieties, folic acid (FA) as a cancer cell-targeting motif and Dexamethasone (DEX) as a nuclear localization signaling molecule, into the protocells. As a result, the FA enabled the modified targeting protocells to deliver the DNA into the cancer cells with an increased efficiency and the DEX promoted the DNA to translocate to cell nuclei, eventually leading to the increased chromosome insertion efficiency of the SB transposon. In vivo study strongly suggested that the transfection efficiency of FA-modified protocells in the tumor tissue was much higher than that in other tissues, which was consistent with the in vitro results. Our studies implied that with the targeting ligand modification, the protocells could be utilized as an efficient targeting gene carrier. Since the protocells were made of neutral lipids without cationic charges, the cytotoxicity of protocells was significantly lower than that of traditional cationic gene carriers such as cationic liposomes and polyethylenimine, enabling the protocells to be employed in a wider dosage range in gene therapy. Our work shows that the protocells are a promising gene carrier for future clinical applications.

Self-assembled organic nanorods for dual chemo-photodynamic therapies

“…Lyso-tracker Red was employed as a probe to conrm the lysosome colocalization and escape of Co-NRs from lysosome. [49][50][51][52][53][54][55][56] As shown in Fig. 3A, aer incubation with Co-NRs for 2 h, HeLa cells were stained with Lyso-tracker Red at 37 C for 30 min.…”

Sleeping Beauty Transposon-Mediated Asparaginase Gene Delivery by a Nanoparticle Platform

“…Recently, Ma et al . improved the efficiency of a SB/carrier system by loading in situ PCR-amplified Sleeping Beauty transposon 36 .…”

“…1). The SB system could efficiently integrate the target gene into the host chromosome for long-term expression both in vitro and in vivo 31–36 . Prior to this study, our lab has successfully accomplished the transient gene delivery using MSN into induced pluripotent stem cells for specific cell-oriented differentiation 37,38 .…”

Sleeping Beauty transposon vectors for therapeutic applications: advances and challenges

“…These LBNs are chemically modulated to present synthetically reiterated MSC-targeting peptides on their surface [69]. Nanoparticle-like protocells with SB encapsulated inside can deliver the therapeutic gene into cancer cells, when folic acid is incorporated as a cancer cell-targeting motif [70]. Furthermore, special hepatocyte-targeted carriers, such as proteoliposomes containing galactose-terminated glycoproteins (e.g., the F protein of the Sendai virus) were demonstrated to effectively deliver the SB cargo into hepatocytes [71].…”