Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells.
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.
Ebeling
Peter
P
Department of Internal Medicine (Cancer Research), University of Duisburg-Essen Medical School, Hufelandstrasse 55, 45122 Essen, Germany. peter.ebeling@uni-essen.de
Bach
P
P
Sorg
U
U
Schneider
A
A
Trarbach
T
T
Dilloo
D
D
Hanenberg
H
H
Niesert
S
S
Seeber
S
S
Moritz
T
T
Flasshove
M
M
D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D036101:Cord Blood Stem Cell Transplantation; D017931:DNA Primers / Q000737:chemistry; D005240:Feasibility Studies; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006085:Graft Survival / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D016511:Severe Combined Immunodeficiency / Q000276:immunology / Q000628:therapy*; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D014161:Transduction, Genetic
D018952:Antigens, CD34; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D017493:Leukocyte Common Antigens