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A widely used third-generation lentiviral packaging system produces virus with enhanced biosafety by eliminating HIV accessory genes and separating packaging elements into three different plasmids. However, for certain vectors such as pLKO.1, third-generation safety features reduce lentiviral titers due to the lack of the accessory gene tat. Here we present a way to improve virus production and target gene knockdown with a modified pLKO.1 CMV pLKO.1C) vector and optimized packaging construct ratios. Replacing the pLKO.1 RSV promoter with the Cytomegalovirus promoter yielded an average of threefold higher titer than standard pLKO.1 packaged using the third-generation system, while optimizing the packaging vector ratios further increased titer and yielded an average of tenfold higher titer than pLKO.1 packaged with the second-generation system.