Abstract
Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts. To examine the copy number effect on the gene expression levels and its stability for a long-term culture for a future application in gene therapy, we constructed a HAC vector carrying the human factor VIII (FVIII) complementary DNA, FVIII-HAC in Chinese hamster ovary (CHO) cells. One and more copies of FVIII gene on the HAC were expressed in the copy-number-dependent manner in the CHO cells. The HAC with 16 copies of FVIII, FVIII (16)-HAC, was transferred from CHO hybrids into a human immortalized mesenchymal stem cell using microcell-mediated chromosome transfer. The expression levels of HAC-derived FVIII transgene products were compared with transfected FVIII plasmids. The former showed expression levels consistent with those of the original clones, even after 50 population doublings, whereas the latter showed a remarkable decrease in expression despite unvarying DNA content, indicating that the gene on the HAC is resistant to gene silencing. These results suggest that the HAC-mediated therapeutic gene-expression system may be a powerful tool for stable expression of transgenes, and possibly for industrial production of gene products.
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Acknowledgements
We wish to thank Drs M Okabe (Osaka University) for providing pCX-EGFP; G Felsenfeld, (National Institutes of Health) for providing pJC5-4. This study was supported in part by a Health and Labor Science Research Grant for Research on HIV/AIDS and Research on Intractable Diseases from the Japanese Ministry of Health, Labour and Welfare, a Grant-in-Aid for Research Activity Start-up, and a Regional Innovation Cluster Program grant (accelerative support) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. All animal studies were approved by the Institutional Animal Care and Use Committee of Tottori University.
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Kurosaki, H., Hiratsuka, M., Imaoka, N. et al. Integration-free and stable expression of FVIII using a human artificial chromosome. J Hum Genet 56, 727–733 (2011). https://doi.org/10.1038/jhg.2011.88
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DOI: https://doi.org/10.1038/jhg.2011.88
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