In vivo expansion of transduced murine hematopoietic cells with a selective amplifier gene.
BACKGROUND
Hematopoietic stem-cell-directed gene transfer has achieved limited success in transducing clinically relevant levels of target cells. The expansion of gene-modified cells is one way to circumvent the problem of inefficient transduction with current vectors. To this end, we have developed 'selective amplifier genes' (SAGs) that encode chimeric proteins that are a fusion of granulocyte colony-stimulating factor receptor and the steroid-binding domain. Prototype SAGs conferred estrogen-responsive growth on murine hematopoietic progenitors.


METHODS
We constructed a retroviral vector coexpressing an SAG for 4-hydroxytamoxifen (Tm)-specific proliferation and the enhanced green fluorescent protein (EGFP). Murine bone marrow cells were transduced with this vector and transplanted into myeloablated mice. Subsequently, recipients were challenged with Tm, and EGFP(+) cells were enumerated.


RESULTS
The challenge induced a significant increase in EGFP(+) leukocytes (21 +/- 4% to 27 +/- 5%), while EGFP(+) cells decreased in untreated animals (21 +/- 5% to 10 +/- 3%). Three months later, bone marrow cells were transplanted from the unchallenged mice to secondary hosts. Again the administration of Tm resulted in an increase of EGFP(+) cells (16 +/- 4% to 35 +/- 3%), contrasting to a decrease in controls (22 +/- 4% to 12 +/- 4%), and the difference was significant for more than 3 months. A detailed study of lineage showed a preferential expansion of EGFP(+) cells in granulocytes and monocytes following Tm administration.


CONCLUSIONS
Long-term repopulating cells were transduced with the SAG, and the transduced granulocyte/monocyte precursors were most likely to be expandable in vivo upon Tm stimulation.
Kume
Akihiro
A
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi 329-0498, Japan.
Koremoto
Masahide
M
Xu
Ruifang
R
Okada
Takashi
T
Mizukami
Hiroaki
H
Hanazono
Yutaka
Y
Hasegawa
Mamoru
M
Ozawa
Keiya
K
D000818:Animals; D016026:Bone Marrow Transplantation / Q000502:physiology; D002455:Cell Division / Q000235:genetics* / Q000502:physiology; D005809:Genes, Regulator / Q000235:genetics* / Q000502:physiology; D017930:Genes, Reporter; D008040:Genetic Linkage; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D051379:Mice; D012190:Retroviridae; D014161:Transduction, Genetic