Figure 1
Figure 1. In vivo transduction of mobilized HSPCs with a first-generation Ad5/35++ vector after mobilization. (A) hCD46 expression on BM total MNCs, lineage-depleted BM cells (Lin−), and LSK cells from hCD46tg mice. Representative hCD46 flow cytometry analysis with MNC (solid black) and LSK cells (dashed black) (left). The gray curve represents MNCs stained with an isotype-matched control. hCD46 MFI on MNCs, Lin−, and LSK cells (right). N = 3. *P < .05, after one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. (B) Mobilization of LSK cells in hCD46tg mice by SC G-CSF injection for 4 days, followed by a single SC injection of AMD3100 on day 5. Forty minutes after the AMD3100 injection, PBMCs were harvested and analyzed by flow cytometry for LSK cells. Representative plots of nonmobilized and mobilized mice are shown. (C) Analysis of HSPC mobilization based on CFU formation. PBMCs were collected before onset of mobilization treatment, before injection of AMD3100, 40 minutes and 3 hours after the AMD3100 injection, as well as on days 3, 7, and 14 after mobilization. The collected cells were subjected to CFU assays and colonies were enumerated 12 days after plating. Shown are mean ± standard deviation (SD), colonies normalized to a blood volume of 100 μL. N = 3. (D) A total of 4 × 1010 vp of the first-generation Ad5/35++-GFP vector was IV injected 40 minutes after AMD3100. To alleviate release of pro-inflammatory cytokines associated with IV Ad vector injection, animals received dexamethasone (10 mg/kg) IP 16 hours and 2 hours before virus injection. Early transduction was analyzed by harvesting PBMCs, BM, and spleen cells at 2 hours after virus injection, and culturing them for 48 hours to allow for GFP expression. Shown is the percentage of GFP+ cells in the LSK cell fractions (analyzed by flow cytometry). N = 3. (E) Animals were mobilized and injected with Ad5/35++-GFP as before. Animals were euthanized and PBMCs collected at 2 hours and 6 hours after transduction. The cells were cultured for 48 hours to allow for GFP expression. Shown is the percentage of GFP+ LSK cells. In addition, GFP expression in peripheral blood LSK cells was analyzed at 3 days after transduction, without culturing of the cells. Unmobilized, untransduced animals were used as controls (mock). N = 3. (F) Animals were mobilized and injected with Ad5/35++-GFP as before. Transduction was analyzed by harvesting BM and splenic cells at day 3, 7, and 14 after Ad5/35++-GFP injection. Nonmobilized control animals were euthanized 2 days after infection. Shown is the percentage of GFP+ cells within total MNCs, and LSK cells in the BM and spleen. N = 3. Values represent means ± SD. *P < .05; **P < .01; ***P < .001; ****P < .0001, after unpaired Student t test comparing nonmobilized controls with animals euthanized 3 days after transduction. dpi, days postinfusion; Iso, isolated; MFI, mean fluorescence intensity; Mobil, mobilized; n.s., not significant.

In vivo transduction of mobilized HSPCs with a first-generation Ad5/35++ vector after mobilization. (A) hCD46 expression on BM total MNCs, lineage-depleted BM cells (Lin−), and LSK cells from hCD46tg mice. Representative hCD46 flow cytometry analysis with MNC (solid black) and LSK cells (dashed black) (left). The gray curve represents MNCs stained with an isotype-matched control. hCD46 MFI on MNCs, Lin−, and LSK cells (right). N = 3. *P < .05, after one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. (B) Mobilization of LSK cells in hCD46tg mice by SC G-CSF injection for 4 days, followed by a single SC injection of AMD3100 on day 5. Forty minutes after the AMD3100 injection, PBMCs were harvested and analyzed by flow cytometry for LSK cells. Representative plots of nonmobilized and mobilized mice are shown. (C) Analysis of HSPC mobilization based on CFU formation. PBMCs were collected before onset of mobilization treatment, before injection of AMD3100, 40 minutes and 3 hours after the AMD3100 injection, as well as on days 3, 7, and 14 after mobilization. The collected cells were subjected to CFU assays and colonies were enumerated 12 days after plating. Shown are mean ± standard deviation (SD), colonies normalized to a blood volume of 100 μL. N = 3. (D) A total of 4 × 1010 vp of the first-generation Ad5/35++-GFP vector was IV injected 40 minutes after AMD3100. To alleviate release of pro-inflammatory cytokines associated with IV Ad vector injection, animals received dexamethasone (10 mg/kg) IP 16 hours and 2 hours before virus injection. Early transduction was analyzed by harvesting PBMCs, BM, and spleen cells at 2 hours after virus injection, and culturing them for 48 hours to allow for GFP expression. Shown is the percentage of GFP+ cells in the LSK cell fractions (analyzed by flow cytometry). N = 3. (E) Animals were mobilized and injected with Ad5/35++-GFP as before. Animals were euthanized and PBMCs collected at 2 hours and 6 hours after transduction. The cells were cultured for 48 hours to allow for GFP expression. Shown is the percentage of GFP+ LSK cells. In addition, GFP expression in peripheral blood LSK cells was analyzed at 3 days after transduction, without culturing of the cells. Unmobilized, untransduced animals were used as controls (mock). N = 3. (F) Animals were mobilized and injected with Ad5/35++-GFP as before. Transduction was analyzed by harvesting BM and splenic cells at day 3, 7, and 14 after Ad5/35++-GFP injection. Nonmobilized control animals were euthanized 2 days after infection. Shown is the percentage of GFP+ cells within total MNCs, and LSK cells in the BM and spleen. N = 3. Values represent means ± SD. *P < .05; **P < .01; ***P < .001; ****P < .0001, after unpaired Student t test comparing nonmobilized controls with animals euthanized 3 days after transduction. dpi, days postinfusion; Iso, isolated; MFI, mean fluorescence intensity; Mobil, mobilized; n.s., not significant.

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