One concern of non-viral gene therapies is the potential limitation on the size of the DNA construct used in formulating an effective therapeutic. Some open reading frames, such as the coding sequence of the cystic fibrosis transmembrane regulator (4.4 kbp), are quite large and, when combined with other required regulatory elements, result in a therapeutic construct of significant size (> 8 kbp). Gene transfer optimization studies are usually performed using reporter gene constructs. Typical reporter genes have much smaller open reading frames (luciferase 1.7 kbp, green fluorescent protein 0.7 kbp) than many therapeutic genes. Therefore, we wanted to determine if our strategies for gene therapy of multiple inherited and acquired diseases will be limited in vivo by the required size of the plasmid DNA payload. To address this aim, we constructed three luciferase reporter plasmids of increasing size—~5, 10, and 20 kbp—using lambda DNA stuffer fragments inserted into the 5 kbp plasmid. These plasmids were compacted to DNA nanoparticles with a 10 kDa polyethylene glycol (PEG) substituted 30-mer lysine polymer containing either trifluoroacetate or acetate counterions at the time of DNA mixing. As evaluated by transmission electron microscopy, these formulations generate compacted DNA that appears as either ellipsoids or rods, respectively. These formulations were delivered to the lungs of Balb/C mice via the intranasal route, and the lungs were harvested and luciferase activity evaluated 2 days after gene transfer. All DNA nanoparticles tested—~5 kbp to 20 kbp plasmids formulated using PEG-lysine polymers with either counterion—had luciferase reporter activities of ~104 RLU/mg protein/pmol DNA above background. Within each formulation group, the reporter gene activity was equivalent (no statistically significant differences) among all three compacted plasmid DNAs. These results indicate that the size of the plasmid used, up to at least 20 kbp, should not be a limitation for lung gene transfer using this non-viral gene therapy platform.