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A number of lentiviral vector systems have been developed for gene delivery and therapy by eliminating and/or modifying viral genetic elements. However, all lentiviral vector systems derived from HIV-1 must have a viral packaging signal sequence, Psi (Ψ), which is placed downstream of 5′ long terminal repeat in a transgene plasmid to effectively package and deliver transgene mRNA. In this study, we examined feasible regions or sequences around Psi that could be manipulated to further modify the packaging sequence. Surprisingly, we found that the sequences immediately upstream of the Psi are highly refractory to any modification and resulted in transgene vectors with very poor gene transduction efficiency. Analysis around the Psi region revealed that there are a few sites that can be used for manipulation of the Psi sequence without disturbing the virus production as well as the efficiency of transgene RNA packaging and gene transduction. By exploiting this new vector system, we investigated the requirement of each of four individual stem-loops of the Psi sequence by deletion mapping analysis and found that all stem-loops, including the SL4 region, are needed for efficient transgene RNA packaging and gene delivery. These results suggest a possible frame of the lentiviral vector that might be useful for further modifying the region/sequence around the packaging sequence as well as directly on the Psi sequence without destroying transduction efficiency.

The Determination of Importance of Sequences Neighboring the Psi Sequence in Lentiviral Vector Transduction and Packaging Efficiency