Abstract
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.
Highlights
A number of lentiviral vector systems have been developed for gene delivery and therapy, as they have an advantage of delivering genes of interest efficiently into non-dividing cells compared to the conventional retroviral vector system [1,2,3]
The third generation of lentiviral vector system was developed as a so-called self-inactivating viral vector, which delivers and expresses only the intended gene of interest, and reported to be unable to generate a full-length viral mRNA once integrated in a target cell chromosome due to the elimination of a region of transcriptional element U3 from the 39-end of the long terminal repeat (LTR) [11,12]
The individual plasmids were transiently transfected into 293FT cells with helper plasmids and the resulting viruses were harvested after 30 hr. pLenti/EGFP with intact Psi sequence and pLenti/Primer Binding Site (PBS).Map, which does not have a Psi sequence as described previously [17], were subjected to the same analysis as controls
Summary
A number of lentiviral vector systems have been developed for gene delivery and therapy, as they have an advantage of delivering genes of interest efficiently into non-dividing cells compared to the conventional retroviral vector system [1,2,3]. Black triangles around the packaging sequence Psi indicate nucleotide positions in which restriction enzyme sites were created to generate lentiviral transgene vectors used in this study.
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