Transfection of airway epithelium by stable PEGylated poly-l-lysine DNA nanoparticles in vivo

Abstract

DNA can be compacted using polyethylene glycol-substituted poly-L-lysine into discrete unimolecular (with respect to DNA) nanoparticles with minor diameter < 20 nm that are stable in normal saline for at least 23 months at 4 degrees C. We compared the activity of firefly luciferase in lungs of C57BL/6 mice that received 100 microg compacted plasmid in 25 microl saline (shown to be the optimal dose) via intratracheal or intranasal instillation with levels in animals given 100 microg naked plasmid or in untreated mice. Mice dosed with compacted DNA nanoparticles had peak activity of luciferase in lung at 2 days postinstillation, which declined in log-linear fashion with a half-life of 1.4 days. Luciferase activity in animals dosed with naked DNA was 200-fold less. Addition of polyethylene glycol to the complex was necessary for efficient gene transfer and animals that received DNA compacted with unmodified poly-L-lysine did not exhibit luciferase activity above background. Immunohistochemical staining for bacterial beta-galactosidase 2 days after administration of a compacted lacZ expression plasmid (n = 8) revealed expression predominantly in the dependent portions of the right lungs of mice, in alveolar and airway epithelial cells, though macrophages and sometimes endothelial cells also were transfected. No staining for beta-galactosidase was observed in uninjected animals (n = 4) or those dosed with naked lacZ plasmid (n = 7). Tissue survey for transgene expression shows expression only in lung and trachea following intranasal administration. Stable compacted DNA nanoparticles transfer exogenous genes to airway epithelium and show promise for lung gene therapy.

Fatty acid–spermine conjugates as DNA carriers for nonviral in vivo gene delivery

“…[36][37][38] The most successful cases occur after intranasal or intratracheal administration, when targeting airway epithelial cells. 3,5,9,39 Tissues containing nondividing cells (as in muscle) or slow mitotic cells (as in the liver) are very attractive targets because pDNA can remain for a long time inside the cells. 40 In relation to the relatively fast kinetics seen here, we do not exclude the possibility that the decrease in expression after 5-7 days may be indicative of toxicity.…”

“…For this purpose, DNA condensation is a recurrent requisite for this type of nonviral therapeutic approach. 5,[8][9][10][11][12] Bloomfield 13 has defined DNA condensation as the collapse of extended DNA chains into compact, orderly particles containing only one or a few molecules. Typically, this process results from a neutralization of the negative charges of the DNA phosphate groups with transition into the ordered phase that occurs when 90% of charges are neutralized.…”

Mechanism of efficient transfection of the nasal airway epithelium by hypotonic shock

“…35 The luciferase expression achieved by our simple nonviral system approaches that of recombinant Sendai virus in the nose 36 and is comparable to that achieved with pDNA/PEI and pDNA/CK30PEG10K. 37 Unlike viral vectors, pDNA alone is not proinflammatory, especially when the CpG dinucleotides are eliminated. 38 It could therefore be repeatedly administered in the airway.…”

Defining strategies to extend duration of gene expression from targeted compacted DNA vectors

“…The development and characterization of such second-generation complexes is in progress. 24 Also, our data suggest that re-engineering the plasmid DNA construct to eliminate promoters prone to silencing (eg, by substitution of a eukaryotic promoter) might well extend the duration of expression. Antibody responses to the complexes, when they occur, were low titer and did not prevent subsequent transfection with sec-R-targeted complexes.…”