181. Quantitative Evaluation of Long Term Gene Expression In Vivo after Delivery of Polyethylenimine-Conjugated Sleeping Beauty Transposon DNA

Many applications of gene therapy require long-lasting expression. In mice, hydrodynamic delivery directs DNA to the liver where when integrated expression can last the lifetime of the animal. Because this form of delivery will be invasive in larger animals, we have been seeking alternative methods of delivery, including condensing DNA with polyethelenimine (PEI). PEI has been reported to deliver genes to the lung with expression lasting for up to 3 months (Belur et al, Molecular Therapy 8: 501|[ndash]|507; 2003). However, using standard hydrodynamic delivery as a |[ldquo]|gold standard|[rdquo]| for achievable levels of gene expression in mice, we have found that simple PEI condensation results in expression of delivered genes at levels that are 10 to more than 100-fold lower than with hydrodynamic delivery. Following injection into the tail vein of PEI-complexed plasmids containing an SB transposon with a luciferase gene, DNA expression was most pronounced in the lung one day post-injection. By 7 days post-injection, expression was most evident in the liver rather than the lung as visualized with a Xenogen|[trade]| in vivo imaging system. JetPEI|[trade]| at N/P ratios of 7 and 9 led to expression levels that ranged from 1|[ndash]|22 |[times]| 106 photons/second (p/s) in the lung 1-day post-injection. However, by day-7 the ranges were from 2|[ndash]|80 |[times]| 104 to 2|[ndash]|9 |[times]| 104 p/s for N/P ratios of 7 and 9, respectively. Because particle sizes should be 100 nm or less for uptake by endocytosis, we sized our PEI-plasmid particles by dynamic laser light scattering. Particles ranged in size from 80|[ndash]|100 nm in 5% dextrose at an N/P of 7 with JetPEI|[trade]|. Because uncomplexed, free PEI is cytotoxic to cells, we hypothesize that the removal of free PEI will permit higher N/P ratios, which might provide increased levels of gene delivery and expression. Consequently, we are testing methods for removal of free PEI so that PEI transposon complexes prepared at higher N/P ratios can be tested for improved levels of long-term expression and animal survival after in vivo delivery.