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.