Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles

Abstract

Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect nondividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting. Gene Therapy (2006) The structural features of gene transfer complexes can be optimized to facilitate efficient gene transfer in vivo. We have developed nanoparticles consisting of single molecules of plasmid DNA condensed with polyethylene glycol (PEG)-substituted lysine peptides.1 The volume, shape and size dimensions of these DNA nanoparticles depend on several parameters, including plasmid size and the lysine counterion present at the time of DNA mixing.2 For example, lysine polymers containing trifluoroacetate and acetate counterions result in the formation of ellipsoidal and rod-like nanoparticles, with the volume of these complexes closely predicted by the partial specific volumes of the constituent components. 1,2For ellipsoidal nanoparticles, microinjection studies indicated a size limitation for nuclear access and transgene expression, with nanoparticles having a minor diameter approaching 25 nm having decreased efficiency. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids. In contrast, the diameter of rod-like nanoparticles ranges between 8 and 11 nm for a series of plasmid sizes, whereas the length of the rod is linearly proportional to the plasmid molecular weight. Because the nuclear membrane pore diameter is approximately 25 nm, these data suggested a size dimension that may limit nuclear transit in these microinjection studies. For intrapulmonary delivery of DNA nanoparticles in mice, efficient gene transfer has been observed for larger plasmids using rod-like formulations. For example, up to 50% of lung cells ar...

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