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A 16-bp RBE element mediated Rep-dependent site-specific integration in AAVS1 transgenic mice for expression of hFIX

Gene Therapy volume 16, pages 589–595 (2009)Cite this article

Abstract

We earlier reported that, a 16 bp Rep-binding element (RBE) was sufficient for mediating Rep-dependent integration into AAVS1 in vitro. We explored here the potential use of this RBE in site-specific genome integration at the AAVS1 site in vivo using transgenic mice carrying the human AAVS1 locus in their genome. In the presence of a Rep-donor plasmid, an human blood coagulation factor IX (hFIX) expression plasmid (pRBE–CMV–hFIX) containing the 16 bp RBE was delivered to AAVS1 transgenic mice by hydrodynamic injection. Insertion of the transgene into the AAVS1 site of the mouse genome was confirmed by nested PCR at the junction of the plasmid/AAVS1 locus. Sequencing analysis found the site-specific insertion in four of seven animals injected with pRBE–CMV–hFIX but in none of the mice injected with pN2–CMV–hFIX, the control construct without the 16 bp RBE or with pRBE–CMV–hFIX plasmid but without co-expressing Rep. Plasma hFIX levels in pRBE–CMV–hFIX-injected animals were higher and lasted longer than in the pN2–CMV–hFIX control group. The levels of hFIX in pRBE–CMV–hFIX-injected animals were also significantly higher than in the control animals after partial hepatectomy (PH). These results showed that the 16 bp RBE could mediate the delivery of a therapeutic gene into the AAVS1 locus in a Rep-dependent, site-specific manner in vivo, suggesting its potential application in gene therapy.

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Abbreviations

AAV:

adeno-associated virus

ITR:

inverted terminal repeat

RBE:

Rep-binding element

hFIX:

human blood coagulation factor IX.

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Acknowledgements

The authors acknowledge the support of the State Hightech Development Program (863-2006AA02Z161; 2007AA021002) and the National Basic Research Program (973.2004CB518803) of China.

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Author notes

  1. Z X Xu and J Z Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, School of Pharmaceutical Science, China Pharmaceutical University, Nanjing, China

    Z X Xu & Q J Wang

  2. State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China

    Z X Xu, J Z Chen, Y B Yue, J Q Zhang, Z H Li, D M Feng, Z C Ruan, L Tian, J L Xue & W Jia

  3. Department of Surgery, University of British Columbia, Vancouver, Canada

    W Jia

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  1. Z X Xu
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Correspondence to Q J Wang or W Jia.

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Xu, Z., Chen, J., Yue, Y. et al. A 16-bp RBE element mediated Rep-dependent site-specific integration in AAVS1 transgenic mice for expression of hFIX. Gene Ther 16, 589–595 (2009). https://doi.org/10.1038/gt.2009.9

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