The central DNA flap increases the transduction efficiency of human CD34⁺ and CD34⁺CD38⁻ CB cells by lentiviral vectors.

(A) Vector constructs. The TRIP vector was derived from the previously described HR vector18 by the insertion, immediately upstream of the internal CMV promoter, of a central HIV-1 DNA fragment encompassing the cPPT and CTS cis-active sequences. cPPT and CTS cis-active sequences are responsible for the formation of the DNA flap during HIV-1 reverse transcription.13 15 Both vectors encode EGFP. (B) Dose-response transduction experiments with TRIP (▪, ▴) and HR (■, ▵) vectors. CD34⁺ CB cells (▪, ■) or CD34⁺CD38⁻ cells (▴, ▵) from the same CB were exposed to vector particles for 24 hours in serum-free medium in the presence of cytokines, and after an additional 48 hours of culture, EGFP expression was analyzed by flow cytometry (“Materials and methods”). The upper panel shows the percentage of CD34⁺EGFP⁺ cells, and the middle panel shows the MFI of the CD34⁺EGFP⁺ cell population. The lower panel shows the EGFP fluorescence activity of the CD34⁺EGFP⁺ cell population (percentage of CD34⁺EGFP⁺ cells multiplied by the MFI, arbitrary units). All are plotted as a function of vector concentration normalized according to P24 levels. The data shown are the results of one representative experiment out of 3 independent experiments.