title: Heat-regulated production and secretion of insulin from a human artificial chromosome vector.
abstract: Human artificial chromosomes (HACs) behave as independent minichromosomes and are potentially useful as a way to achieve safe, long-term expression of a transgene. In this study, we sought to elucidate the potential of HAC vectors carrying the human proinsulin transgene for gene therapy of insulin-dependent diabetes mellitus (IDDM) using non-beta-cells as a host for the vector. To facilitate the production of mature insulin in non-beta-cells and to safely regulate the level of transgene expression, we introduced furin-cleavable sites into the proinsulin coding region and utilized the heat shock protein 70 (Hsp70) promoter. We used Cre-loxP-mediated recombination to introduce the gene cassettes onto 21DeltapqHAC, a HAC vector whose structure is completely defined, present in human fibrosarcoma HT1080 cells. We observed long-term expression and stable retention of the transgene without aberrant translocation of the HAC constructs. As expected, the Hsp70 promoter allowed us to regulate gene expression with temperature, and the production and secretion of intermediates of mature insulin were made possible by the furin-cleavable sites we had introduced into proinsulin. This study can be an initial step on the application of HAC vectors on the gene delivery to non-beta-cells, which might provide a direction for future treatment for diabetes.
authors.affiliation: Department of Human Genome Science, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan.
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Suda
authors.forename: Motonobu
authors.initials: M
authors.lastname: Katoh
authors.forename: Masaharu
authors.initials: M
authors.lastname: Hiratsuka
authors.forename: Masato
authors.initials: M
authors.lastname: Takiguchi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Inoue
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
mesh_terms: D045744:Cell Line, Tumor; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D005354:Fibrosarcoma / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D011401:Promoter Regions, Genetic / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D007328:Insulin