Inhibition of GM-CSF receptor function by stable RNA interference in a NOD/SCID mouse hematopoietic stem cell transplantation model.
RNA interference (RNAi) describes a highly conserved mechanism of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA (dsRNA). Whereas RNAi is applied to study gene function in different organisms and in variant cell types, little is known about RNAi in human hematopoietic stem and progenitor cells and their myeloid progeny. To address this issue, short hairpin RNAs (shRNA) were designed to target the common beta-chain of the human receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (betaGMR). These receptors regulate proliferation, survival, differentiation, and functional activity of hematopoietic cells. In addition to markedly inhibiting mRNA and protein expression, anti-beta-GMR shRNAs were also found to inhibit receptor function in a cell culture model. Furthermore, lentiviral gene transfer of shRNA expression cassettes into primary normal CD34+ cells selectively inhibited colony formation of transduced progenitors when stimulated with GM-CSF/IL-3 but not when stimulated with cytokines that do not signal via beta-GMR. Finally, anti-beta-GMR shRNAs had no detectable effect on engraftment or lineage composition of lentivirally transduced human CD34+ cells transplanted into NOD/SCID mice. However, the growth defect of transduced colony-forming cells under stimulation with GM-CSF/IL-3 remains unchanged in bone marrow cells harvested from individual NOD/SCID mice 6 weeks after transplantation. These data indicate that lentiviral gene transfer of shRNA expression cassettes may be used to induce long-term RNAi in human hematopoietic stem and progenitor cells for functional genetics and potential therapeutic intervention.
Hannover Medical School, Department of Hematology and Oncology, Hannover, Germany. M.Scherr@t-online.de
Michaela
M
Scherr
Karin
K
Battmer
Iris
I
Dallmann
Arnold
A
Ganser
Matthias
M
Eder
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002455:Cell Division; D002470:Cell Survival; D017931:DNA Primers; D020868:Gene Silencing / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics*; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D018123:Receptors, Interleukin / Q000235:genetics; D016185:Receptors, Interleukin-3 / Q000235:genetics; D053649:Receptors, Interleukin-5; D033581:Stem Cell Transplantation / Q000379:methods*; D014162:Transfection / Q000379:methods; D014183:Transplantation, Heterologous
D017931:DNA Primers; D034741:RNA, Small Interfering; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D018123:Receptors, Interleukin; D016185:Receptors, Interleukin-3; D053649:Receptors, Interleukin-5