title: Integration-free and stable expression of FVIII using a human artificial chromosome.
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.
authors.affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Kurosaki
authors.forename: Masaharu
authors.initials: M
authors.lastname: Hiratsuka
authors.forename: Natsuko
authors.initials: N
authors.lastname: Imaoka
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Iida
authors.forename: Narumi
authors.initials: N
authors.lastname: Uno
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.forename: Chie
authors.initials: C
authors.lastname: Ishihara
authors.forename: Yuwna
authors.initials: Y
authors.lastname: Yakura
authors.forename: Jun
authors.initials: J
authors.lastname: Mimuro
authors.forename: Youichi
authors.initials: Y
authors.lastname: Sakata
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeya
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism*; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics
chemical_list: D005169:Factor VIII