Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells.
Keywords
Abstract
OBJECTIVE
Although gene transfer with retroviral vectors has shown distinct clinical success in defined settings, efficient genetic manipulation of hematopoietic progenitor cells remains a challenge. To address this issue we have evaluated different transduction protocols and retroviral constructs in the non-obese diabetes (NOD)/severe combined immunodeficiency disease (SCID) xenograft model.
METHODS
An extended transduction protocol requiring 144 h of in vitro manipulation was compared to a more conventional protocol requiring 96 h only.
RESULTS
While pretransplantation analysis of cells transduced with a retroviral vector, expressing the enhanced green fluorescent protein (EGFP) marker gene, demonstrated significantly higher overall transduction rates for the extended protocol (33.6 +/- 2.3 vs. 22.1 +/- 3.8%), EGFP expression in CD34+ cells before transplantation (4.0 +/- 0.9 vs. 11.6 +/- 2.5%), engraftment of human cells in NOD/SCID bone marrow 4 weeks after transplantation (4.5 +/- 1.7 vs. 36.5 +/- 9.4%) and EGFP expression in these cells (0 +/- 0 vs. 11.3 +/- 2.8%) were significantly impaired. When the 96 h protocol was used in combination with the spleen focus forming virus (SFFV)/murine embryonic stem cell (MESV) hybrid vector SFbeta11-EGFP, high transduction rates for CD45+ (41.0 +/- 5.3%) and CD34+ (38.5 +/- 3.7%) cells prior to transplantation, as well as efficient human cell engraftment in NOD/SCID mice 4 weeks after transplantation (32.4 +/- 3.5%), was detected. Transgene expression was observed in B-lymphoid (15.9 +/- 2.0%), myeloid (36.5 +/- 3.5%) and CD34+ cells (10.1 +/- 1.5%).
CONCLUSIONS
Our data show that CD34+ cells maintained in cytokines for multiple days may differentiate and loose their capacity to contribute to the haematological reconstitution of NOD/SCID mice. In addition, the SFFV/MESV hybrid vector SFbeta11-EGFP allows efficient transduction of and gene expression in haematopoietic progenitor cells.
