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Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state

Abstract

We have shown previously that transgene expression can be suppressed in hematopoietic cells using vectors that are responsive to microRNA (miRNA) regulation. Here we investigate the potential of this approach for more sophisticated control of transgene expression. Analysis of the relationship between miRNA expression levels and target mRNA suppression suggested that suppression depends on a threshold miRNA concentration. Using this information, we generated vectors that rapidly adjust transgene expression in response to changes in miRNA expression. These vectors sharply segregated transgene expression between closely related states of therapeutically relevant cells, including dendritic cells, hematopoietic and embryonic stem cells, and their progeny, allowing positive/negative selection according to the cells' differentiation state. Moreover, two miRNA target sites were combined to restrict transgene expression to a specific cell type in the liver. Notably, the vectors did not detectably perturb endogenous miRNA expression or regulation of natural targets. The properties of miRNA-regulated vectors should allow for safer and more effective therapeutic applications.

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Figure 1: Examining the relationship between miRNA expression and target suppression.
Figure 2: Endogenous miRNA regulation is not perturbed by miRNA-regulated LV.
Figure 3: Combinatorial arrangements of miRNA target sites can be used to achieve desired expression profiles in vivo.
Figure 4: Intrinsic differences in miRNA expression can be exploited to restrict transgene expression in mature dendritic cells and whole hematopoietic lineages.
Figure 5: Achieving differentiation state–specific transgene expression in human ES cells and their progeny.
Figure 6: Making TK/ganciclovir-mediated suicide conditional on miRNA expression.

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Acknowledgements

HUES were kindly provided by Doug Melton from Harvard Stem Cell Institute (Cambridge, MA, USA), under specific Material Transfer Agreement to C.G. We are grateful to Irene Bozzoni, Desiree Bonci and Roger Tsien for providing reagents, Silvia Gregori and Daniela Tomasoni for human monocytes, Angelo Lombardo, Michele De Palma and Roberta Mazzieri for helpful discussions, and Lucia Sergi Sergi and Giulia Schira for technical help. We would also like to thank the San Raffaele Centre of Statistics for Biomedical Sciences for suggestions on data analysis. This work was supported by grants from Telethon (TIGET grant), EU (Projects LSHB-CT-2004-005276, RIGHT and LSHB-CT-2004-005242, CONSERT) and the Italian Ministry of Scientific Research to L.N., and EU Grant LSB-CT-2004-503257 to G.L. B.D.B. is the recipient of a Natural Science and Engineering Research Council of Canada (NSERC) fellowship. B.G. is the recipient of a research fellowship from the German Research Foundation (DFG Forschungsstipendium).

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B.D.B. designed and performed research and analyzed data concerning vector design, miRNA regulation and dendritic cells, and wrote the paper. B.G. designed and performed research and analyzed data on HS cells and differentiation state-specific and suicide vectors, and wrote the paper. A.C. performed research and analyzed data concerning vector design and miRNA regulation. S.C. and G.L. performed research and analyzed data regarding the ES cell studies. M.A., A.Z. and A.B. performed research. C.G. coordinated ES cell work. L.N. coordinated the project, designed research, analyzed data and wrote the paper.

Corresponding author

Correspondence to Luigi Naldini.

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Brown, B., Gentner, B., Cantore, A. et al. Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat Biotechnol 25, 1457–1467 (2007). https://doi.org/10.1038/nbt1372

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