Repopulation of NOD/SCID mice by transduced cord blood CD34+ cells: analysis of engraftment and gene-transfer efficiency
| Experimental group . | In vitro analysis . | In vivo analysis . | ||||
|---|---|---|---|---|---|---|
| % EGFP expression . | % TMTX-R progenitors . | % Human CD45+(range) . | % EGFP+ (range)* . | Human progenitors . | ||
| Trimetrexate resistant (%) . | PCR+colonies (%) . | |||||
| Expanded CD34+ cells (n = 10)† | 0 | 0 | 13.5 ± 17 | 0 | 0/228 (0) | 0/20 (0) |
| (2.18-54.5) | ||||||
| AM/MGirL22Y-transduced CD34+ cells (n = 9)‡ | 18 -34 | 6.5 -8 | 15.6 ± 16 | 0.2 ± 0.1 | 0/118 (0) | 0/50 (0) |
| (1.0-53) | (0-0.6) | |||||
| RD114/MGirL22Y preload only at 24 h (n = 9) | 75 -87 | 58 -75 | 3.5 ± 5 | 20.0 ± 22 | 24/106 (23) | 30/61 (49) |
| (0.6-15.8) | (0.6-71) | |||||
| RD114/MGirL22Y preload only at 48 h (n = 6) | 50 -69 | 45 -66 | 8.7 ± 8 | 40.5 ± 40 | 81/326 (25) | 58/98 (59) |
| (1.7-23.5) | (1.8-92) | |||||
| Experimental group . | In vitro analysis . | In vivo analysis . | ||||
|---|---|---|---|---|---|---|
| % EGFP expression . | % TMTX-R progenitors . | % Human CD45+(range) . | % EGFP+ (range)* . | Human progenitors . | ||
| Trimetrexate resistant (%) . | PCR+colonies (%) . | |||||
| Expanded CD34+ cells (n = 10)† | 0 | 0 | 13.5 ± 17 | 0 | 0/228 (0) | 0/20 (0) |
| (2.18-54.5) | ||||||
| AM/MGirL22Y-transduced CD34+ cells (n = 9)‡ | 18 -34 | 6.5 -8 | 15.6 ± 16 | 0.2 ± 0.1 | 0/118 (0) | 0/50 (0) |
| (1.0-53) | (0-0.6) | |||||
| RD114/MGirL22Y preload only at 24 h (n = 9) | 75 -87 | 58 -75 | 3.5 ± 5 | 20.0 ± 22 | 24/106 (23) | 30/61 (49) |
| (0.6-15.8) | (0.6-71) | |||||
| RD114/MGirL22Y preload only at 48 h (n = 6) | 50 -69 | 45 -66 | 8.7 ± 8 | 40.5 ± 40 | 81/326 (25) | 58/98 (59) |
| (1.7-23.5) | (1.8-92) | |||||
Umbilical cord blood CD34+ cells were assayed for transduction efficiency based on EGFP expression and trimetrexate-resistant (TMTX-R) progenitors before injection into NOD/SCID recipients. After 8 to 10 weeks, the animals were sacrificed and bone marrow was analyzed for human engraftment (CD45+), EGFP expression, and TMTX-R progenitors. Unselected human progenitors from engrafted animals were probed by PCR for the proviral genome. CD34+ cells were efficiently transduced by RD114/MGirL22Y particles preloaded onto retronectin-coated plates after 24 to 48 hours of prestimulation. There was a significant (P < .05) loss of human engraftment compared with controls in the mice that received CD34+ cells transduced after 24 hours in culture by RD114/MGirL22Y particles. The decrease in engraftment was not significant compared with controls in the animals that received CD34+ cells transduced at 48 hours in culture with RD114/MGirL22Y particles. There was significant (P < .05) functional marking based on EGFP expression and overall gene transfer based on PCR analysis in both groups of mice that received CD34+ cells transduced with RD114/MGirL22Y particles compared with mice that received CD34+ cells transduced with AM/MGirL22Y particles.
Percent EGFP expression of engrafted human mononuclear cells.
n = number of murine recipients of 1.0 to 1.5 × 105cells that exhibited more than 0.5% human CD45+ cells.
Transduction by preloading alone at 24 hours (n = 3) or with the addition of conditioned medium containing vector particles added at 48 and 72 hours.