Figure 2
Figure 2. Stable in vivo HSPC transduction with integrating HD-Ad5/35++ vectors. (A) Vector genome structure. The transposon vector (HD-Ad–GFP (left) carries an Ef1α-driven GFP expression cassette that is flanked by inverted transposon repeats and frt sites. The second vector (HD-Ad–SB) (right) provides both Flpe recombinase and SB100× transposase in trans. Both are HD vectors containing the affinity-enhanced Ad35++ fiber knob. (B) Experimental design of the study demonstrating in vivo HSPC transduction with HD-Ad vectors. hCD46tg mice were mobilized and IV injected with HD-Ad–GFP (2 injections, each 2 × 1010 vp) or a 1:1 mixture of HD-Ad–GFP plus HD-Ad–SB (2 injections, each 4 × 1010 vp). Groups of mice were euthanized at 3 days, 4, 8, 12, and 20 weeks after injection, and BM cells, splenocytes, and PBMCs were harvested. GFP expression in total MNCs (for BM, spleen, and PBMC) and BM LSK cells was analyzed by flow cytometry. (C) Mobilized and nonmobilized hCD46tg animals were injected with HD-Ad–SB + HD-Ad–GFP. BM cells (left), splenocytes (middle), and PBMCs (right) were collected 3 days after transduction, and expression of GFP in different lineages as well as LSK cells and total MNCs were analyzed via flow cytometry. N = 5. *P < .05 after two-way ANOVA with Bonferroni posttesting.**P < .01; ***P < .001; ****P < .0001. (D) GFP expression in total BM (upper left), spleen (lower left), and peripheral blood MNCs (lower right), as well as BM LSK cells (upper right). Circles represent animals injected with HD-Ad–GFP only (N = 6). Squares represent animals injected with HD-Ad–GFP + HD-Ad–SB euthanized at 3 days (N = 5), 4 (N = 10), 8 (N = 10), 12 (N = 11), and 20 weeks (N = 5) after transduction. Each data point represents a single animal. **P < .01. (E) GFP marking in hematopoietic lineages of BM and spleen. Animals were euthanized 3 days as well as 8 and 20 weeks after transduction, and GFP expression in different lineages was analyzed via flow cytometry. Shown is the mean ± SD percentage of GFP+ cells in the indicated lineages. ****P < .0001. Some of the data, eg, the decrease in GFP+/CD19+ cells and the increase in GFP+/Gr-1+ cells in the BM between weeks 8 and 20 cannot be readily explained. ITR, inverted terminal repeats; PGK, phosphoglycerate kinase.

Stable in vivo HSPC transduction with integrating HD-Ad5/35++ vectors. (A) Vector genome structure. The transposon vector (HD-Ad–GFP (left) carries an Ef1α-driven GFP expression cassette that is flanked by inverted transposon repeats and frt sites. The second vector (HD-Ad–SB) (right) provides both Flpe recombinase and SB100× transposase in trans. Both are HD vectors containing the affinity-enhanced Ad35++ fiber knob. (B) Experimental design of the study demonstrating in vivo HSPC transduction with HD-Ad vectors. hCD46tg mice were mobilized and IV injected with HD-Ad–GFP (2 injections, each 2 × 1010 vp) or a 1:1 mixture of HD-Ad–GFP plus HD-Ad–SB (2 injections, each 4 × 1010 vp). Groups of mice were euthanized at 3 days, 4, 8, 12, and 20 weeks after injection, and BM cells, splenocytes, and PBMCs were harvested. GFP expression in total MNCs (for BM, spleen, and PBMC) and BM LSK cells was analyzed by flow cytometry. (C) Mobilized and nonmobilized hCD46tg animals were injected with HD-Ad–SB + HD-Ad–GFP. BM cells (left), splenocytes (middle), and PBMCs (right) were collected 3 days after transduction, and expression of GFP in different lineages as well as LSK cells and total MNCs were analyzed via flow cytometry. N = 5. *P < .05 after two-way ANOVA with Bonferroni posttesting.**P < .01; ***P < .001; ****P < .0001. (D) GFP expression in total BM (upper left), spleen (lower left), and peripheral blood MNCs (lower right), as well as BM LSK cells (upper right). Circles represent animals injected with HD-Ad–GFP only (N = 6). Squares represent animals injected with HD-Ad–GFP + HD-Ad–SB euthanized at 3 days (N = 5), 4 (N = 10), 8 (N = 10), 12 (N = 11), and 20 weeks (N = 5) after transduction. Each data point represents a single animal. **P < .01. (E) GFP marking in hematopoietic lineages of BM and spleen. Animals were euthanized 3 days as well as 8 and 20 weeks after transduction, and GFP expression in different lineages was analyzed via flow cytometry. Shown is the mean ± SD percentage of GFP+ cells in the indicated lineages. ****P < .0001. Some of the data, eg, the decrease in GFP+/CD19+ cells and the increase in GFP+/Gr-1+ cells in the BM between weeks 8 and 20 cannot be readily explained. ITR, inverted terminal repeats; PGK, phosphoglycerate kinase.

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