742. Human Artificial Chromosome (HAC) Vector Provides Long-Term Therapeutic Transgene Expression in Normal Human Primary Fibroblasts

Abstract

Top of pageAbstract Human artificial chromosomes (HACs) are segregating freely from host chromosomes through a set of cell divisions. Accordingly, the host genome is not disrupted and the expression of the transgene could be sustained for a prolonged period without suffering the effects of its surrounding sequence on the host genome. These are the ideal properties required for vectors in gene therapy, and therefore HACs are potentially useful to ensure both safety and duration of gene expression in therapeutic gene delivery. We constructed a novel human chromosome 21-derived HAC (21|[Delta]|pqHAC) vector, which devoid of most expressed genes by telomere truncation at the genomic regions proximal to the centromere in both p and q arms, and inserted human erythropoietin (EPO) gene, which is a growth factor for erythroid cells and widely used for the clinical treatment of anemia in renal insufficiency, into the HAC (EPO-21|[Delta]|pqHAC) as a model of therapeutic transgenes. Although low transfer efficiency of intact HACs to the cells has hampered the studies using normal human primary cells, the major targets for ex vivo gene therapy, we successfully demonstrated the introduction of EPO-21|[Delta]|pqHAC vector into normal primary human fibroblasts (hPFs) with micro cell-mediated chromosome transfer (MMCT). However, the mitotic stability of EPO-21|[Delta]|pqHAC vector in normal hPFs and the growth ability of the resultant hPF clones to obtain graft cells for transplantation remained to be elucidated. Here, we studied the hPF clones harboring the EPO-21|[Delta]|pqHAC vector cytogenetically and demonstrated that the structurally defined, extra artificial chromosome was highly maintained in normal hPFs under non-selective condition at a constant copy number mostly in a single copy per cell without accompanying aberration of host chromosomes. Mitotic-loss rates were determined, and the level of stability of EPO-21|[Delta]|pqHAC vector was comparable with those reported for the other top-down and bottom-up artificial chromosomes in human fibro-sarcoma cell line HT1080 cells. These hPF clones proliferated from a single colony up to 3.8x107 cells and then their growth stopped, suggesting that cell senescence occurred and the introduction of the EPO-21|[Delta]|pqHAC vector did not cause immortalization. We also showed the long-term expression of human epo gene in the hPFs carrying the EPO-21|[Delta]|pqHAC vector. In conclusion, this study shows the potential application of the 21|[Delta]|pqHAC vector in hormone-replacement gene therapy, which might ensure safety with no lesion of host chromosomes and provides long-term therapeutic transgene expression. This also implies that utilization of the HAC vector offers novel options for ex vivo gene therapy.