Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells

Li,Shu‑Jun; Luo,Ying; Zhang,Le‑Meng; Yang,Wei; Zhang,Guo‑Gang

doi:10.3892/mmr.2017.6131

Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells

Authors:
- Shu‑Jun Li
- Ying Luo
- Le‑Meng Zhang
- Wei Yang
- Guo‑Gang Zhang
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Affiliations: Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China, Department of Geriatric Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China, Department of Thoracic Medicine, Hunan Cancer Hospital Affiliated to Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China, Department of Respiratory Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410000, P.R. China, Department of Cardiovascular Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410000, P.R. China
Published online on: January 19, 2017 https://doi.org/10.3892/mmr.2017.6131
Pages: 1313-1318
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Hemophilia B occurs due to a deficiency in human blood coagulation factor IX (hFIX). Currently, no effective treatment for hemophilia B has been identified, and gene therapy has been considered the most appropriate treatment. Mesenchymal stem cells (MSCs) have homing abilities and low immunogenicity, and therefore they may be potential cell carriers for targeted drug delivery to lesional tissues. The present study constructed an adeno‑associated virus integration site 1 (AAVS1)‑targeted vector termed AAVS1‑green fluorescent protein (GFP)‑hFIX and a zinc finger nuclease (ZFN) expression vector. Nucleofection was used to co‑transfect the targeting vector and the ZFN expression vector into human MSCs. The GFP‑positive cells were selected using flow cytometry. Site‑specific integration clones were obtained following the monoclonal culture, subsequent detections were performed using polymerase chain reaction and Southern blotting. Following the confirmation of stem cell traits of the site‑specific integration MSCs, the in vivo and in vitro expression levels of hFIX were detected. The results demonstrated that the hFIX gene was successfully transfected into the AAVS1 locus in human MSCs. The clones with the site‑specific integration retained stem cell traits of the MSCs. In addition, hFIX was effectively expressed in vivo and in vitro. No significant differences in expression levels were identified among the individual clones. In conclusion, the present study demonstrated that the exogenous gene hFIX was effectively expressed following site‑specific targeting into the AAVS1 locus in MSCs; therefore, MSCs may be used as potential cell carriers for gene therapy of hemophilia B.

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