Technical Report | Published:

Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer

Nature Medicine volume 12, pages 585591 (2006) | Download Citation

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Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidence indicates that miRNAs have distinct expression profiles and play crucial roles in numerous cellular processes, although the extent of miRNA regulation is not well known. By challenging mice with lentiviral vectors encoding target sequences of endogenous miRNAs, we show the efficiency of miRNAs in sharply segregating gene expression among different tissues. Transgene expression from vectors incorporating target sequences for mir-142-3p was effectively suppressed in intravascular and extravascular hematopoietic lineages, whereas expression was maintained in nonhematopoietic cells. This expression profile, which could not be attained until now, enabled stable gene transfer in immunocompetent mice, thus overcoming a major hurdle to successful gene therapy. Our results provide novel in situ evidence of miRNA regulation and demonstrate a new paradigm in vector design with applications for genetic engineering and therapeutic gene transfer.

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References

  1. 1.

    , , & Identification of novel genes coding for small expressed RNAs. Science 294, 853–858 (2001).

  2. 2.

    & MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531 (2004).

  3. 3.

    & Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA 11, 241–247 (2005).

  4. 4.

    et al. Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739 (2002).

  5. 5.

    , , & MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86 (2004).

  6. 6.

    et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005).

  7. 7.

    , & Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 436, 214–220 (2005).

  8. 8.

    et al. MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat. Genet. 36, 1079–1083 (2004).

  9. 9.

    , & Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4, 346–358 (2003).

  10. 10.

    Gene transfer for hemophilia: can therapeutic efficacy in large animals be safely translated to patients? J. Thromb. Haemost. 3, 1682–1691 (2005).

  11. 11.

    & Dangerous liaisons: the role of “danger” signals in the immune response to gene therapy. Blood 100, 1133–1140 (2002).

  12. 12.

    , & Humoral immune response in mice against a circulating antigen induced by adenoviral transfer is strictly dependent on expression in antigen-presenting cells. Blood 101, 2551–2556 (2003).

  13. 13.

    et al. Helper-dependent adenoviral vectors mediate therapeutic factor VIII expression for several months with minimal accompanying toxicity in a canine model of severe hemophilia A. Blood 103, 804–810 (2004).

  14. 14.

    et al. Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice. Blood 103, 3700–3709 (2004).

  15. 15.

    et al. Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer. J. Clin. Invest. 111, 1347–1356 (2003).

  16. 16.

    et al. Factors influencing therapeutic efficacy and the host immune response to helper-dependent adenoviral gene therapy in hemophilia A mice. J. Thromb. Haemost. 2, 111–118 (2004).

  17. 17.

    et al. Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells. Blood 105, 2307–2315 (2005).

  18. 18.

    & Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat. Rev. Genet. 5, 396–400 (2004).

  19. 19.

    , & siRNAs can function as miRNAs. Genes Dev. 17, 438–442 (2003).

  20. 20.

    , & Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333 (2002).

  21. 21.

    , , , & Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat. Biotechnol. 23, 108–116 (2005).

  22. 22.

    , & The three-dimensional structure of human splenic white pulp compartments. J. Histochem. Cytochem. 51, 655–664 (2003).

  23. 23.

    et al. Immune response to green fluorescent protein: implications for gene therapy. Gene Ther. 6, 1305–1312 (1999).

  24. 24.

    , , & Principles of microRNA-target recognition. PLoS Biol. 3, e85 (2005).

  25. 25.

    et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906 (2000).

  26. 26.

    et al. A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis. Cell 123, 819–831 (2005).

  27. 27.

    et al. The widespread impact of mammalian microRNAs on mRNA repression and evolution. Science 310, 1817–1821 (2005).

  28. 28.

    et al. Liver sinusoidal endothelial cells tolerize T cells across MHC barriers in mice. J. Immunol. 175, 139–146 (2005).

  29. 29.

    , , , & miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34, D140–D144 (2006).

  30. 30.

    , , , & Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

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Acknowledgements

We thank E. Allievi and I. Benzoni (San Raffaele Conditional Mutagenesis Core Facility) for help in generating transgenic mice; B. Cullen for providing the miR-30aT, E. Hauben for human monocytes, F. Sanvito for histology, A. Cantore for technical assistance, and M. De Palma, A. Lombardo and D. Lillicrap for discussions. This work was supported by grants from Telethon (TIGET grant), the European Union (Projects LSHB-CT-2004-005276, RIGHT and LSHB-CT-2004-005242, CONSERT) and the Italian Ministry of Scientific Research (to L.N.). B.D.B. is the recipient of a Natural Science and Engineering Research Council of Canada fellowship.

Author information

Affiliations

  1. San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), via Olgettina 58, 20132 Milano, Italy.

    • Brian D Brown
    • , Mary Anna Venneri
    • , Anna Zingale
    • , Lucia Sergi Sergi
    •  & Luigi Naldini
  2. Vita Salute San Raffaele University, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy.

    • Luigi Naldini

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Luigi Naldini.

Supplementary information

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  1. 1.

    Supplementary Fig. 1

    Analysis of transgene expression in the peripheral blood of F1 TgN.PGK.GFP.142-3pT mice.

  2. 2.

    Supplementary Fig. 2

    mir-142-3p sharply segregates gene expression between hematopoietic and nonhematopoietic lineages in the spleen of transgenic mice.

  3. 3.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nm1398

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