Introduction of the green fluorescent protein gene into hematopoietic stem cells results in prolonged discrepancy of in vivo transduction levels between bone marrow progenitors and peripheral blood cells in nonhuman primates.
BACKGROUND
The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning.


METHODS
Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34(+) cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy x 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells.


RESULTS
Although 13-37% of transduced cells contained the GFP provirus and 11-13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5-10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes.


CONCLUSIONS
Low levels of GFP-transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo.
Hanazono
Yutaka
Y
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan. hanazono@jichi.ac.jp
Terao
Keiji
K
Shibata
Hiroaki
H
Nagashima
Takeyuki
T
Ageyama
Naohide
N
Asano
Takayuki
T
Ueda
Yasuji
Y
Kato
Ikunoshin
I
Kume
Akihiro
A
Hasegawa
Mamoru
M
Ozawa
Keiya
K
D000818:Animals; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics*; D008252:Macaca fascicularis; D008297:Male; D014161:Transduction, Genetic / Q000379:methods
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins