Figure 1.
Caraphenol A enhances LV gene marking in HeLa and primary hematopoietic cells. (A) Chemical structure of caraphenol A, α-viniferin, and resveratrol. (B) HeLa cells were transduced with pRRL-SIN-MND-EGFP (termed LV) at a MOI of 10, in the presence of DMSO (diluent control = no compound) or indicated concentrations of resveratrol (closed orange circles), α-viniferin (closed purple circles), or caraphenol A (closed green circles) over the course of 8 hours, before removal of caraphenol A and LV and then ex vivo culture. Cells were analyzed 5 days later by flow cytometry for EGFP expression (n = 3 independent experiments). (C) HeLa cells were transduced as earlier, with various MOIs of LV in the presence of 30 μM of each compound or DMSO (closed blue circles) for 8 hours and analyzed as earlier (n = 3). Data are shown as linear plots (mean ± standard deviation [SD]). *P < .032, **P < .0021, ****P < 0002, ****P < .0001 by 2-tailed Student t test comparing percentage EGFP expression in caraphenol A- and DMSO-treated cells. (D) LV transduction of CD34+ human UCB (n = 6 donors), human granulocyte colony-stimulating factor mobilized peripheral blood (mPB) (n = 6 donors), and nonhuman primate bone marrow aspirate (n = 2 donors) cells. CD34+ cells were transduced with LV (MOI = 8) in the absence or presence of 30 μM caraphenol A for 20 hours before LV and compound removal and expansion. Cells were analyzed 7 days later by flow cytometry for EGFP expression. Data presented as dot plots (mean ± SD) *P < .0406, ****P < .0001 by 2-tailed Student t test. (E) Average VCN in UCB-CD34+ (n = 3 donors) at increasing MOI of LV treatment with either DMSO vehicle control or caraphenol A at 30 μM. VCN was calculated as a ratio of copy number of integrated LV Gag sequences per RNAse P copies. Data presented as dot plots (mean ± SD). ****P < .0001 by 2-tailed Student t test. (F) Effects of caraphenol A (10 μM, open green circles or 30 μM, closed green circles), resveratrol (30 μM, closed orange circles), and rapamycin (20 μg/mL, closed red circles) on proliferation of mPB-CD34+ cells compared with DMSO (0.06%, closed blue circles; n = 3 donors, n = 2 for DMSO). Proliferation of UCB-CD34+ cells after compound treatment are shown in supplemental Figure 2A. Data presented as dot plots (mean ± SD). (G) VCN in mPB-CD34+ cells (n = 4 donors) of 2 separate batches of clinical-grade CL204i-EF1α-hγc-OPT SIN-lentiviral vectors (VP5609 and VP5610) developed for the treatment of SCID-X1, incorporating an internal EF1α promoter for human interleukin 2Rγc. Cell were transduced with SCID-X1 or LV at an MOI = 15 in the presence of DMSO, 30 μM caraphenol A, 20 μg/mL rapamycin, or 10 μM PGE-2 (closed light blue circles). Data presented as mean ± SD. **P = .0024, ****P < .0001 by Student t test, comparing DMSO with caraphenol A.

Caraphenol A enhances LV gene marking in HeLa and primary hematopoietic cells. (A) Chemical structure of caraphenol A, α-viniferin, and resveratrol. (B) HeLa cells were transduced with pRRL-SIN-MND-EGFP (termed LV) at a MOI of 10, in the presence of DMSO (diluent control = no compound) or indicated concentrations of resveratrol (closed orange circles), α-viniferin (closed purple circles), or caraphenol A (closed green circles) over the course of 8 hours, before removal of caraphenol A and LV and then ex vivo culture. Cells were analyzed 5 days later by flow cytometry for EGFP expression (n = 3 independent experiments). (C) HeLa cells were transduced as earlier, with various MOIs of LV in the presence of 30 μM of each compound or DMSO (closed blue circles) for 8 hours and analyzed as earlier (n = 3). Data are shown as linear plots (mean ± standard deviation [SD]). *P < .032, **P < .0021, ****P < 0002, ****P < .0001 by 2-tailed Student t test comparing percentage EGFP expression in caraphenol A- and DMSO-treated cells. (D) LV transduction of CD34+ human UCB (n = 6 donors), human granulocyte colony-stimulating factor mobilized peripheral blood (mPB) (n = 6 donors), and nonhuman primate bone marrow aspirate (n = 2 donors) cells. CD34+ cells were transduced with LV (MOI = 8) in the absence or presence of 30 μM caraphenol A for 20 hours before LV and compound removal and expansion. Cells were analyzed 7 days later by flow cytometry for EGFP expression. Data presented as dot plots (mean ± SD) *P < .0406, ****P < .0001 by 2-tailed Student t test. (E) Average VCN in UCB-CD34+ (n = 3 donors) at increasing MOI of LV treatment with either DMSO vehicle control or caraphenol A at 30 μM. VCN was calculated as a ratio of copy number of integrated LV Gag sequences per RNAse P copies. Data presented as dot plots (mean ± SD). ****P < .0001 by 2-tailed Student t test. (F) Effects of caraphenol A (10 μM, open green circles or 30 μM, closed green circles), resveratrol (30 μM, closed orange circles), and rapamycin (20 μg/mL, closed red circles) on proliferation of mPB-CD34+ cells compared with DMSO (0.06%, closed blue circles; n = 3 donors, n = 2 for DMSO). Proliferation of UCB-CD34+ cells after compound treatment are shown in supplemental Figure 2A. Data presented as dot plots (mean ± SD). (G) VCN in mPB-CD34+ cells (n = 4 donors) of 2 separate batches of clinical-grade CL204i-EF1α-hγc-OPT SIN-lentiviral vectors (VP5609 and VP5610) developed for the treatment of SCID-X1, incorporating an internal EF1α promoter for human interleukin 2Rγc. Cell were transduced with SCID-X1 or LV at an MOI = 15 in the presence of DMSO, 30 μM caraphenol A, 20 μg/mL rapamycin, or 10 μM PGE-2 (closed light blue circles). Data presented as mean ± SD. **P = .0024, ****P < .0001 by Student t test, comparing DMSO with caraphenol A.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal