Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences

Abstract

Gene-transfer vectors based on lentiviruses are distinguished by their ability to transduce non-dividing cells1,2. The HIV-1 proteins Matrix, Vpr and Integrase have been implicated in the nuclear import of the viral genome in non-dividing cells3,4,5. Here we show that a sequence within pol is also required in cis. It contains structural elements previously associated with the progress of reverse transcription in target cells6,7,8,9. We restored these elements in cis within late-generation lentiviral vectors10,11. The new vector transduced to a much higher efficiency several types of human primary cells, when both growing and growth-arrested, including haematopoietic stem cells assayed by long-term repopulation of NOD/SCID mice. On in vivo administration into SCID mice, the vector induced higher plasma levels of human clotting factor IX (F.IX) than non-modified vector. Our results indicate that nuclear translocation of the genome is a rate-limiting step in lentiviral infection of both dividing and non-dividing cells, and that it depends on protein and nucleic acid sequence determinants. Full rescue of this step in lentivirus-based vectors improves performance for gene-therapy applications.

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Figure 1: Insertion of sequences from pol of HIV-1 into the backbone of a lentiviral vector enhances the kinetics of transgene expression and the efficiency of transduction.
Figure 2: The cPPT sequence does not affect encapsidation or reverse transcription of vector RNA, but enhances nuclear translocation of the transcripts within target cells.
Figure 3: Transduction of human primary cells is improved by the cPPT vector.
Figure 4: Efficient transduction of human haematopoietic cells, including NOD/SCID repopulating cells, by the cPPT vector.
Figure 5: Improved performance of cPPT(s) vector in vivo.

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Acknowledgements

We thank W. Piacibello, F. Bussolino and members of their laboratories for help in setting up experiments with haematopoietic cells and endothelial cells, respectively; and T. Dull, M. Kelly and M. Gasmi for providing constructs. The financial support of Telethon–Italy (grant A.143 to L.N.) and MURST (Grant 9905313431 to LN) is gratefully acknowledged.

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Correspondence to Luigi Naldini.

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