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Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters

Abstract

Transferring multiple genes into the same cell allows for the combination of genetic correction, marking, selection and conditional elimination of transduced cells or the reconstitution of multisubunit components and synergistic pathways. However, this cannot be reliably accomplished by current gene transfer technologies. Based on the finding that some cellular promoters intrinsically promote divergent transcription, we have developed synthetic bidirectional promoters that mediate coordinate transcription of two mRNAs in a ubiquitous or a tissue-specific manner. Lentiviral vectors incorporating the new promoters enabled efficient dual gene transfer in several tissues in vivo after direct delivery or transgenesis, and in a human gene therapy model. Because divergent gene pairs, likely transcribed from shared promoters, are common in the genome, the synthetic promoters that we developed may mimic a well-represented feature of transcription. Vectors incorporating these promoters should increase the power of gene function studies and expand the reach and safety of gene therapy.

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Figure 1: Gene transfer performance of bidirectional lentiviral vectors.
Figure 2: Tissue-specific expression by bidirectional LVs.
Figure 3: Comparison of dual gene transfer by bidirectional and bicistronic lentiviral vectors.
Figure 4: Ex vivo and in vivo dual-gene transfer and therapy by bidirectional vectors.

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Acknowledgements

We are grateful to R. Loew, A.D. Simmons, D. Vignali, C. Lois and M. Sampaolesi for providing plasmids, A. Lombardo, F. Santoni De Sio, C. Scielzo, E. Bongarzone and J. Gonzalez for help with some experiments, F. Benedicenti and L. Sergi Sergi for technical help and M. De Palma for critical reading of the manuscript. This research was supported by grants from Telethon (TIGET), EU (QLK3-1999-00859 and QLRT-2001-02114), AIRC, and the Italian Ministry of Scientific Research to L.N. M.A. is a recipient of a scholarship award from O.N.A.O.S.I.

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Correspondence to Luigi Naldini.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Relationship between transgene expression and the number of integrated vector copies (PDF 751 kb)

Supplementary Fig. 2

Gene transfer performance of IRES-based bicistronic lentiviral vectors (PDF 837 kb)

Supplementary Fig. 3

Blood analysis of mice transplant with purified (lin-) murine bone marrow progenitors (PDF 551 kb)

Supplementary Fig. 4

Dual transgenesis by bidirectional LVs (PDF 832 kb)

Supplementary Notes (PDF 70 kb)

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Amendola, M., Venneri, M., Biffi, A. et al. Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol 23, 108–116 (2005). https://doi.org/10.1038/nbt1049

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