1. Volume 2, Issue 1, Pages 47-55 (July 2000)
Development of a Novel Trans-Lentiviral Vector That Affords Predictable Safety 
Xiaoyun Wu, John K. Wakefield, Hongmei Liu, Hongling Xiao, Robert Kralovics, Josef T. Prchal, John C. Kappes 
Molecular Therapy 
Volume 2, Issue 1, Pages (July 2000)
DOI: /mthe
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Genetic components of the trans-lentiviral vector system. The trans-lentiviral packaging construct is illustrated as pCMV-gag-proΔvpr. The pLR2P-vpr-RT-IN construct encodes the Vpr-RT-IN fusion protein, which is packaged into the Gag/Gag-Pro particles and provides the reverse transcriptase and integrase function. Proteolytic processing by the viral protease liberates the mature RT (p51/p66) and IN proteins (24, 25). The pPC-eGFP gene transfer vector and the pMD.G plasmid are also depicted. Trans-lentiviral vector particles are produced by the transfection of all four genetic elements.
Molecular Therapy 2000 2, 47-55DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

3. FIG. 4
Recombination-dependent lentiviral DNA mobilization assay. (A) Lentiviral vector particles are produced by transient transfection of 293T cells. Through nonspecific mRNA encapsidation, it is possible that a small proportion of the progeny particles contain mRNA derived from the packaging construct in addition to that of the gene transfer vector. To increase the efficiency of infection, the transfection-derived lentiviral vector particles were concentrated by ultracentrifugation (90,000g, 2 h, 4°C). The pellets were resuspended and titrated on HeLa cell monolayers using GFP as a marker. 107 infectious units (IU) were used to transduce 5 × T cells (m.o.i. = 2). (B) Genetic recombination between the mRNAs of the packaging construct and the vector during reverse transcription could generate env minus recombinant lentiviral genomes, such as LTR-Ψ-gag-pol-tat/rev-LTR. If stably integrated into the chromosomes of transduced 293T cells, such recombinant proviral genomes could express viral proteins and produce env minus progeny retrovirus-like particles that encapsidate the recombinant's mRNA genome. To detect these recombinant genomes, the transduced 293T cells were cotransfected with the pDM.G expression plasmid (2 μg) to pseudotype any progeny lentivirus-like particles with the VSV-G protein and pCMV-tat to upregulate expression of recombinant proviral genomes. (C) Three days later, the culture supernatants were concentrated by ultracentrifugation and resuspended in 1 ml of DMEM. Progeny particles with encapsidated recombinant lentiviral genomes are depicted. (D) HeLa-puro cells were then infected with the pseudotyped vector particles. Forty hours after infection, the infected HeLa-puro cells monolayer is trypsinized and replated in medium containing puromycin (5 μg/ml). Pseudotyped particles could confer resistance to puromycin if they contained a recombinant lentivirus genome that was reverse transcribed and integrated and expressed Tat protein. The detection of resistant colonies would indicate mobilization of the recombinant genome (gag transfer).
Molecular Therapy 2000 2, 47-55DOI: ( /mthe )
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4. FIG. 2
Transduction of human macrophages with the trans-lentiviral vector. Peripheral blood monocytes were prepared from healthy human donors and cultured in RPMI containing 10% human serum for 1 day in six-well plates (6 × 106 cells/well). The next day, the cells were washed extensively to remove the nonadherent cells and then cultured for 2 weeks in RPMI containing 10% human AB serum and GMS. The monolayer macrophage cultures were infected (m.o.i. = 5, as determined on monolayer cultures of HeLa cells) with the trans-lenti and lentiviral vectors containing eGFP under control of the CMV promoter. The cells were photographed 5 days later using a fluorescence microscope.
Molecular Therapy 2000 2, 47-55DOI: ( /mthe )
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5. FIG. 3
Transduction of human CD34+ bone marrow cells with the trans-lentiviral vector. (A) CD34+ cells were derived from bone marrow aspirates of health adults by magnetic cell sorting. FACS analysis indicated that the CD34+ cell population was 98% pure. Unstimulated CD34+ cells (2.5 ? 105) were infected for 4 h (m.o.i. = 10) with the lenti and trans-lentiviral vectors containing the GFP trans gene. The infected and uninfected cells were analyzed by flow cytometry for GFP expression after 3 days. In four independent experiments the transduction efficiency of the trans-lentiviral vector ranged from 15 to 43%. (B) GFP expression in differentiated myeloid cells. The transduced CD34+ cells were cultured for 3 days in serum-free suspension culture (as above) and then plated at a final concentration of 5 ? 103 cells/ml in Methocult H-4531 semisolid methylcellulose medium (StemCell Technologies Inc., Vancouver, Canada) in the presence of 3 U/ml EPO. After 14 days of culture, the GFP expression in erythroid (BFU-E), monocytic (CFU-M), and multilineage (CFU-GEMM) progenitor colonies was examined using bright-field (upper panels) and fluorescence microscopy (lower panels).
Molecular Therapy 2000 2, 47-55DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Lentiviral vector recombination-dependent DNA mobilization. Supernatants from cultures of 293T cells that were transduced with 107 infectious units (IU) of the lentiviral and trans-lentiviral vectors, respectively, were collected as described under Materials and Methods and used to infect replica cultures of HeLa-puro cells. One set was infected 2 h after Nevirapine had been added to the culture medium (5 μM). The other set was infected without the addition of Nevirapine. The control culture contained uninfected HeLa-puro cells. After 12 days of selection in medium containing puromycin, the cell monolayers were stained with crystal violet. The number of individual colony-forming units (CFU)/resistant colonies was counted using a light microscope. The actual number of CFUs that were produced from the supernatants of the transduced 293T cell cultures is shown below each culture dish. The results of this experiment are representative of five independent analyses.
Molecular Therapy 2000 2, 47-55DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Genetic analysis of recombinant lentivirus. High-molecular-weight DNA was prepared from puromycin-resistant cells and subjected to PCR using primer pairs specific for sequences of the vector and packaging construct. (A) Sequence analysis of the 5’ end of the recombinant genome. Using a sense primer specific for the U3 region of the 5’ LTR of the gene transfer vector and an antisense primer specific for 3’ gag sequences of the packaging construct, a PCR fragment of approximately 2000 base pairs was amplified and cloned into pUC19. The nucleotide sequence at the junction of the vector and gag gene is illustrated. Of 10 clones analyzed, the sequence was identical. (B) Sequence analysis of the 3’ end of the recombinant genome. Using a sense primer specific for the first tat exon of the packaging construct and an antisense primer specific for the 3’ U3 region of the vector, a DNA fragment of approximately 2500 bp was PCR amplified, cloned into pUC19, and sequenced. In 9 of 9 clones that were analyzed, the 3’ end of the vector was found to be joined with the poly(A) tract of the packaging construct. The nucleotide sequence between the U3 region of the vector and the poly(A) tract of the packaging construct is illustrated. Among the 9 clones, four distinct recombinant genotypes were detected.
Molecular Therapy 2000 2, 47-55DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
The trans-RT-IN prevents DNA mobilization by the trans-lentiviral vector. (A) Replica cultures of 293T cells were infected with 107 infectious units of the trans-lentiviral vector. Recombination between the gag-pro packaging plasmid and the gene transfer vector in the transduced 293T cells could produce an RT-IN minus recombinant. One possible structure of such a recombinant is illustrated. Two days postinfection (p.i.), one of the cultures was transfected with the pMD.G expression plasmid, while the other culture was cotransfected with both pMD.G and the pLR2P-vpr-RT-IN expression plasmid. (B) The supernatants of the 293T cell cultures were collected 3 days later and used to infect HeLa-puro cells. (C) The cell monolayers were stained with crystal violet after 12 days of selection in medium containing puromycin. The number of colony-forming units (CFU) was counted using a light microscope. The actual number of CFUs that were produced from the supernatants of the transduced 293T cell cultures is shown below each culture dish.
Molecular Therapy 2000 2, 47-55DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions