Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer

A Seven-Year Storage Report of Good Manufacturing Practice–Grade Naked Plasmid DNA: Stability, Topology, andIn Vitro/In VivoFunctional Analysis

“…These in vitro and, more importantly, in vivo expression data clearly demonstrate that even very long but controlled stable storage conditions preserve the topology of naked plasmid DNA and ensure effective and reproducible transgene expression. The importance of stable storage conditions was earlier shown for plasmid DNA stored at -80°C for 13 months (Walther, 2003). However, our current study significantly extends the earlier report, demonstrating that, after 7-year storage at -20°C, plasmid DNA topology and its functionality for gene therapy applications are preserved at a very high degree.…”

“…The plasmid pUK21 (PlasmidFactory) was used as a reference standard. CGE analyses were performed using a Beckman P/ACE MDQ instrument equipped with an laser induced fluorescence (LIF) detector (488/520 nm) as described (Schmidt, 1999;Walther, 2003). Briefly, coated capillaries (DB-17; J&W Scientific) with an effective length of 30 cm to the detector window, an inner diameter of 100 lm, and a coating thickness of 0.1 lm were used.…”

“…However, the ability to completely resuspend the DNA is mandatory and is highly dependent on DNA purity. In an earlier study, we have shown that storage temperature rather than the duration of storage is important to preserve plasmid topology (Walther et al, 2003;Voss, 2007).…”

“…The technology of large-scale production of plasmid DNA required for clinical applications has been significantly improved since more than a decade (Schleef and Blaesen, 2009;Schleef et al, 2010). This includes also the progress made in plasmid purification, which is closely associated with improvements in long-term DNA stability (Walther et al, 2003;Schleef and Bleasen, 2009). This progress is paralleled with the development of physical transfer technologies of naked DNA, such as electroporation, particle bombardment, jet-injection, and ultrasound (sonoporation), which are often used to improve transfer efficiencies (AlDosari and Gao, 2009).…”

Cardiovascular function following acute volume overload for hydrodynamic gene delivery to the liver

“…The stock DNA solution was maintained at 41C for a maximum of 2 weeks after thawing, as this has been shown not to affect activity. 18 In some experiments, the DNA was made up in Ringer's solution (0.147 M NaCl, 0.004 M KCl, 0.002 M CaCl 2 ) (Baxter Healthcare Ltd, Norfolk, UK) to minimize electrolyte imbalances following the infusion.…”