Down-regulation of retroviral transgene expression during differentiation of progenitor-derived dendritic cells.
OBJECTIVE
Hematopoietic progenitor cells are a promising source for generation of genetically modified dendritic cells. A prerequisite for using these cells in therapeutic approaches is stable vector-mediated transgene expression during and after cell maturation. We investigated the expression of enhanced green fluorescence protein (EGFP) mediated by retroviral vectors in dendritic cells and other hematopoietic cells differentiated in vitro.


METHODS
CD34(+) cells were efficiently transduced with retroviral vector constructs known to mediate different expression levels due to distinct cis-acting elements. EGFP(+) cells were purified by cell sorting and differentiated to monocytes, granulocytes, dendritic cells, and erythrocytes. Coexpression of EGFP and cell type-specific markers was analyzed by flow cytometry.


RESULTS
Transgene expression from various retroviral vectors was silenced exclusively in dendritic cells, but not in other mature myeloid cells. Loss of EGFP was most pronounced in cells initially displaying low expression levels. This was confirmed by using a retroviral vector coding for a variant of EGFP with significantly reduced half-life. In contrast, a majority of dendritic cells showed stable expression when a self-inactivating retroviral construct using an internal cytomegalovirus promotor was used.


CONCLUSIONS
We suggest that expression from the retroviral long terminal repeat is silenced during dendritic cell differentiation in vitro. High levels of stable transgene product in progenitor cells may mask a loss of expression. An improvement of retroviral vectors mediating stable transgenic expression is necessary for therapeutic approaches using gene-modified dendritic cells.
Lindemann
Carsten
C
EUFETS AG, Idar-Oberstein, Germany.
Schilz
Andrea J
AJ
Emons
Barbara
B
Baum
Christopher
C
Löw
Rainer
R
Fauser
Axel A
AA
Kuehlcke
Klaus
K
Eckert
Hans-Georg
HG
D019070:Cell Lineage / Q000235:genetics*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000502:physiology*; D015536:Down-Regulation / Q000235:genetics; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D009000:Monocytes / Q000166:cytology / Q000502:physiology; D012190:Retroviridae; D014161:Transduction, Genetic