Heat-shrinking DNA nanoparticles for in vivo gene delivery.

The particle size of a PEG-peptide DNA nanoparticle is a key determinant of biodistribution following i.v. dosing. DNA nanoparticles of <100 nm in diameter are sufficiently small to cross through fenestrated endothelial cells to target hepatocytes in the liver. In addition, DNA nanoparticles must be close to charge-neutral to avoid recognition and binding to scavenger receptors found on Kupffer cells and endothelial cells in the liver. In the present study, we demonstrate an approach to heat shrink DNA nanoparticles to reduce their size to <100 nm to target hepatocytes. An optimized protocol heated plasmid DNA at 100 °C for 10 min resulting in partial denaturation. The immediate addition of a polyacridine PEG-peptide followed by cooling to room temperature resulted in heat-shrunken DNA nanoparticles that were ~70 nm in diameter compared with 170 nm when heating was omitted. Heat shrinking resulted in the conversion of supercoiled DNA into open circular to remove strain during compaction. Heat-shrunken DNA nanoparticles were stable to freeze-drying and reconstitution in saline. Hydrodynamic dosing established that 70 nm heat-shrunken DNA nanoparticles efficiently expressed luciferase in mouse liver. Biodistribution studies revealed that 70 nm DNA nanoparticles are rapidly and transiently taken up by liver whereas 170 nm DNA nanoparticles avoid liver uptake due to their larger size. The results provide a new approach to decrease the size of polyacridine PEG-peptide DNA nanoparticles to allow penetration of the fenestrated endothelium of the liver for the purpose of transfecting hepatocytes in vivo.

Metabolically stabilized double-stranded mRNA polyplexes.

The metabolic instability of mRNA currently limits its utility for gene therapy. Compared to plasmid DNA, mRNA is significantly more susceptible to digestion by RNase in the circulation following systemic dosing. To increase mRNA metabolic stability, we hybridized a complementary reverse mRNA with forward mRNA to generate double-stranded mRNA (dsmRNA). RNase A digestion of dsmRNA established a 3000-fold improved metabolic stability compared to single-stranded mRNA (ssmRNA). Formulation of a dsmRNA polyplex using a PEG-peptide further improved the stability by 3000-fold. Hydrodynamic dosing and quantitative bioluminescence imaging of luciferase expression in the liver of mice established the potent transfection efficiency of dsmRNA and dsmRNA polyplexes. However, hybridization of the reverse mRNA against the 5' and 3' UTR of forward mRNA resulted in UTR denaturation and a tenfold loss in expression. Repeat dosing of dsmRNA polyplexes produced an equivalent transient expression, suggesting the lack of an immune response in mice. Co-administration of excess uncapped dsmRNA with a dsmRNA polyplex failed to knock down expression, suggesting that dsmRNA is not a Dicer substrate. Maximal circulatory stability was achieved using a fully complementary dsmRNA polyplex. The results established dsmRNA as a novel metabolically stable and transfection-competent form of mRNA.