Increased non-viral gene transfer levels in mice by concentration of cationic lipid DNA complexes formed under optimized conditions.
BACKGROUND
A number of non-viral gene transfer reagents including cationic lipid DNA complexes (LDC) have been developed and were improved by changing the ratio of their components. To determine the effect of other parameters during complex formation affecting the efficacy of LDC, the conditions during complexation were varied without changing the ratios of the components.


METHODS
LDC were formed at fixed ratios of an equimolar mixture of DOTAP and cholesterol to DNA to the condensing agent protamine sulfate according to different protocols at varying final concentrations. The influence of these parameters on transfection efficiency and physical properties of the complexes was determined.


RESULTS
Changing the order of addition of compounds during complex formation affected the size distribution, the charge of the LDC, the interaction between the lipids and the accessibility of the DNA. At fixed ratios of the components, higher transfection efficiencies were observed with more condensed LDC. Complexation in higher volumes increased transduction efficiency of the complexes after intravenous inoculation. Due to restrictions on the injectable volume, the LDC were formed in the optimal volume and subsequently concentrated by ultrafiltration. The concentrated complexes maintained transduction efficiency and up to 60-fold higher in vivo transduction levels were obtained.


CONCLUSIONS
In addition to the ratio of the components of cationic lipid DNA complexes, the final concentration and the order of addition of compounds during complex formation are critical for high transduction efficiency. Concentration of LDC formed under optimal conditions can be used to further increase in vivo gene transfer levels.
Nchinda
Godwin
G
Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany.
Zschörnig
Olaf
O
Uberla
Klaus
K
D000818:Animals; D002412:Cations / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D018014:Gene Transfer Techniques; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D011479:Protamines / Q000737:chemistry / Q000378:metabolism
D002412:Cations; D008055:Lipids; D008081:Liposomes; D046911:Macromolecular Substances; D011479:Protamines; D004247:DNA