Effective, reproducible, and scalable methods for DNA-lipid assembly are important for the success of non-viral vectors in in vivo gene therapy. We hypothesized DNA-lipid assembly would be optimal if started from a liquid monophase where both DNA and lipids separately form molecular or micellar solutions prior to mixing, without preexisting condensed lipid phases, thus allowing DNA-lipid assembly under conditions close to equilibrium. Previously, we found that mixing plasmid DNA, 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), cholesterol and a cationic lipid, 1, 2-dioleoyl-3-(trimethylammonio) propane (DOTAP) in 50% (v/v) aqueous ethanol spontaneously produced an optically transparent solution. Upon ethanol removal, DNA-lipid nanoparticles (Genospheres) were formed. For comparison with well-known technologies, different DNA-lipid particles were prepared by interaction of plasmid DNA and stable or ethanol-destabilized lipid vesicles by combining the components in water or 30% (v/v) aqueous ethanol, respectively. Among the three studied DNA-lipid assembly methods, only Genospheres combined the properties of small size (less than or around 100 nm), high incorporation of both lipid and DNA, high degree of DNA protection (dye accessibility 5-12%), a narrow distribution of particle density and when immuno-targeted, the highest transfection efficiency in HER2-overexpressing cells in vitro. We conclude that the Genosphere assembly methodology offers advantages for the development of effective, scalable and targetable non-viral gene delivery vectors.