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Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro

Nature Biotechnology volume 24, pages 566–571 (2006)Cite this article

Abstract

RNA interference occurs when cytoplasmic small interfering RNAs (siRNAs) enter the RNA-induced silencing complex and one strand guides cleavage of the target RNA by the Argonaute 2 protein1,2,3. A significant concern when applying siRNAs or expressing small hairpin RNAs (shRNAs) in human cells is activation of the interferon (IFN) response4,5,6,7,8,9,10. Synthetic siRNAs harboring certain motifs can induce an immune response when delivered to mouse and human immune cells such as peripheral blood mononuclear cells, monocytes, plasmacytoid dendritic cells (pDCs) and nonplasmacytoid dendritic cells (mDCs)11,12,13. In the present study we have tested the immunostimulatory effects of lipid-delivered siRNAs versus Pol III promoter–expressed shRNAs in primary CD34+ progenitor–derived hematopoietic cells. We show that in this system, lipid-delivered siRNAs are potent inducers of IFNα and type I IFN gene expression, whereas the same sequences when expressed endogenously are nonimmunostimulatory.

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Figure 1: CD34+ progenitor-derived immune cells produce IFN and IFN-inducible gene expression in response to treatment with IFN or to transfection with polyIC.
Figure 2: Transfection of synthetic siRNA to site I or site II induces IFNα protein production and IFN-inducible gene expression in CD34+ progenitor–derived monocytes.
Figure 3: IFN and IFN-inducible gene expression in CD34+ cells transduced with lentiviruses expressing GFP alone, shRNAs to site I or II and the triple construct containing site I shRNA, TAR decoy and chimeric VA1-CCR5 ribozyme.
Figure 4: Chemokine and cytokine expression profiles in CD34+ progenitors and CD34+ progenitor–derived monocytes.

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Acknowledgements

The authors would like to thank Lucy Brown and Claude Yosballa at the COH FACS Facility for FACS assays and analysis. This work was supported by National Institutes of Health grants AI29329, AI42552, HL07470 and AI061389.

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Author notes

  1. Marjorie A Robbins

    Present address: Protiva Biotherapeutics, Inc., 100-3480 Gilmore Way, Burnaby, British Columbia, V5G 4Y1, Canada

Authors and Affiliations

  1. Division of Molecular Biology, Beckman Research Institute, City of Hope, Duarte, 91010, California, USA

    Marjorie A Robbins, Mingjie Li, Haitang Li & John J Rossi

  2. Biomedical Research Division, Beckman Coulter, Inc., Fullerton, 92834, California, USA

    Irene Leung, Doris V Boyer & Yong Song

  3. Integrated DNA Technologies, Coralville, 52241, Iowa, USA

    Mark A Behlke

  4. Division of Molecular Biology, Graduate School of Biological Sciences, Beckman Research Institute, City of Hope, 1450 E. Duarte Rd., Duarte, 91010, California

    John J Rossi

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Correspondence to John J Rossi.

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Supplementary information

Supplementary Fig. 1

CD34+ progenitor derived immune cells are a mixed population of monocytes and dendritic cells. (PDF 83 kb)

Supplementary Fig. 2

Schematic of HIV-7 vector used to transduce CD34+ progenitor cells with therapeutic RNAs. (PDF 94 kb)

Supplementary Fig. 3

Inhibition of HIV-1 replication in CD34+ cells expressing anti-HIV RNAs. (PDF 60 kb)

Supplementary Fig. 4

CD34+ progenitors expressing GFP only or shRNA or a triple cassette differentiate to monocytes to similar levels. (PDF 91 kb)

Supplementary Fig. 5

Differentiation of control or stably transduced CD34+ progenitor derived immune cells expressing GFP, shII or the triple construct produces a mixed population of monocytes and plasmacytoid dendritic cells. (PDF 75 kb)

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Robbins, M., Li, M., Leung, I. et al. Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro. Nat Biotechnol 24, 566–571 (2006). https://doi.org/10.1038/nbt1206

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