Prolonged organ retention and safety of plasmid DNA administered in polyethylenimine complexes

Abstract

Polyethylenimine (PEI) Recently, polyethylenimine (PEI) has drawn attention as a versatile, inexpensive, and useful nonviral transfection vector. 1 A variety of cell types such as monocytes, 2 dendritic cells, 3 myoblast cells, 4 and hepatocytes 5 were studied as target cells for PEI-mediated gene transfection. In vivo, PEI was shown to be an efficient transfection vector in several organs such as the kidney, liver, and lung. [6][7][8] However, these studies are mostly focused on the expression of the PEI/DNA limited to target organs in vivo or specific cell types in vitro. Although the knowledge of biodistribution fates of plasmid DNA may reveal the feasibility of the cellular level targeting in vivo, there is little understanding of the overall and quantitative organ distribution of plasmid DNA after administration in PEI complexes. Moreover, the safety of PEI/DNA complexes after repeated dosing has not been well documented, despite the potential requirement for repeated administration due to the episomal property of plasmids.In this study, we tested the distribution fate and safety of plasmid DNA after administering DNA in PEI complexes. Quantitative and competitive polymerase chain reaction (PCR) was employed for the assay of plasmid DNA in the biological samples. Here, we report that in comparison to naked DNA, the organ distribution was higher and remarkably prolonged after administration of plasmid DNA in PEI complexes, and that once a week dosing of PEI/DNA complexes over 3 weeks did not alter the histology of major organs such as the liver, lung, and kidney.PEI/DNA complexes with PEI nitrogen to DNA phosphate ratio (N/P ratio) of 10:1 were prepared in 5% glucose using 25 kDa PEI (Aldrich, St Quentin, France). The N/P ratio of 10:1 was chosen since it was shown to efficiently transfect cells in vivo. 9 The levels of plasmid DNA in biological samples were determined using competitive and quantitative PCR. Quantitative PCR is based on co-amplification of the sample template together with various amounts of internal standard (IS) competitor sharing with the target the primer recognition sites, but differing in size. The competitor was constructed to share the same sense and antisense primer used for target amplification. 10 As an example, Figure 1 shows the gel bands and internal standard curves obtained after running competitive PCR of DNA extracts from the liver 6 h after injection of naked DNA or PEI/DNA complexes. The IS for quantitative PCR was the 188-bp deleted mutant of pCMV␤. The construction of the competitor and PCR conditions were described previously. 10 The PCR products were 449 bp and 261 bp for the target and the IS, respectively. The band densities of the target gene PCR product diminished as the amounts of IS increased (Figure 1a and b). The quantitation of gel band density by densitometry generated internal standard curves of high linearity (Figure 1c). The curves also show that we could detect as low as fg-level plasmids by quantitative PCR. The amounts of plasmid DNA in the samples we...

Gene delivery to the lung using protein/polyethylenimine/plasmid complexes

“…Numerous investigations into the mechanisms of plasmid delivery suggest that PEI is taken up by cells with or without DNA, and that larger quantities can be toxic in vitro 23,24 and in vivo. 25 We have observed this in vitro where concentrations of PEI in the range of 80 nmol/ml or greater do show cytotoxic effects, and associated with this toxicity is a decline in gene expression as measured by the percentage of GFP-positive cells or total luciferase per well (Orson, unpublished). While there is no gross toxicity that we have observed in the lung in animals receiving up to 5 g of DNA at an N:P ratio of 15, cells that have taken up excess quantities of the complexes or free PEI could have reduced gene expression as a result of interference with transcriptional or translational pathways, as has been reported in vitro, or from nonspecific toxicity of unbound free PEI.…”

Induction of gp120-specific protective immune responses by genetic vaccination with linear polyethylenimine–plasmid complex

“…A number of PEI molecules have been described with varying molecular sizes and structures-among them, branched PEI with an average molecular weight of 800 kDa (PEI800) or 25 kDa (PEI25), and linear forms with an average molecular weight of 22 kDa (PEI22)-have been widely used and characterized both in vivo and in vitro [26]. In vivo, systemically delivered PEI22-DNA complexes resulted in the transduction of the lung, spleen, liver, heart and kidney, with no histological change observed in these organs [27]. PEI22-DNA complexes can repeatedly be administered to animals without eliciting an immune response against the polymer, although the development of an immune response against the exogenously expressed protein has been reported to reduce the level of protein expression [28].…”

Gene transfer vector biodistribution: pivotal safety studies in clinical gene therapy development

“…Some authors also used an internal control, which is often a competitive piece of DNA used in the same reaction tube. 12,15,[17][18][19][20] The advantage of this technique is a precise control of inhibition in the same tube, the inconvenience being the sensitivity decrease because of competition. We would recommend qPCR in triplicate, with spiking of one of the reaction tubes with a low DNA amount (as in Hanke et al 13 ).…”

“…19 Comparison of aerosolization and i.v. injection of PEI DNA also showed a wide distribution by both routes at early times.…”