991. Construction of a Novel Human Artificial Chromosome and Its Use for Gene Delivery
Authors: Motonobu Katoh, Fumiaki Ayabe, Satoko Norikane, Hidetoshi Hoshiya, Xianying Ren, Tetsuji Suda, Chiga Okita, Mitsuo Oshimura
Journal: Molecular Therapy (2004)

A group of genes on a human chromosome or its fragment (hCF) is transferrable from donor to recipient cells by microcell-mediated chromosome transfer. Since the hCF is autonomously replicated and segregated as an extra chromosome in the host cells, 1) it does not disrupt the genomic context of the host, 2) its copy number is restricted and persistent and 3) up to megabase- sized DNA fragment is transferable. Our previous study revealed that use of engineered hCFs successfully achieved delivery and functional expression of human immunoglobulin genes in mice. But carry over of genes out of interest on hCF is not suitable for therapeutic applications. In addition, chromosome engineering requires substantial steps and it is not practical to prepare hCF panels corresponding broad range of genes. We aimed at constructing a structurally defined human artificial chromosome (HAC) vector into which circular DNA can be inserted by Cre/lox system. We chose the human chromosome 21 (hChr21) for the HAC construction, since sequence information is available from public databases. The hChr21 was transferred to homologous recombination proficient chicken DT40 cells, followed by truncation of distal q-arm by telomere seeding, introduction of loxP sequence by targeted integration and finally truncation of distal q- arm. Removal of q- and p-arm was confirmed by PCR and FISH analysis. These engineered hCFs named 21HAC vectors were transferrable from DT40 to human HT1080 and mouse ES cells, via CHO cells. Long-term culture in the absence of selection revealed that the 21HAC vectors could stably maintained in the HT1080 and mouse ES cells. Site-specific insertion of circular DNA into the loxP site on the 21 HAC vector was tested using GFP gene expression cassette as a reporter in HT1080 cells. Correct insertion was selected by reconstruction of neo gene split on the HAC vector and a circular DNA insert, that confers G418 resistance. Persistent expression of the introduced GFP gene was observed during serial culture in medium without G418, indicating that the loxP site integrated in the pericentromeric region was not heterochromatinized. Mouse ES cells carrying the GFP/21HAC vector was induced neuronal differentiation in vitro. Colonies detected by antibody against neuron-specific -tubulin expressed GFP, suggesting that the presence of the HAC vector did not disturb differentiation of ES cells. These results indicated that the HAC vector can be used for functional study of transgene in human and mouse cells. The 21HAC vectors could be potentially developed as gene delivery vector applicable for ex vivo gene therapy. Prerequisite for a gene delivery vector is stable maintenance in somatic cells and performance of gene expression regulation. To address these issues, following studies are ongoing; 1) tissue specific gene expression in hepatocytes under regulation of albumin enhancer/promoter, 2) induction of tissue specific gene expression during in vitro differentiation of human Mesenchymal Stem Cells and 3) construction of non- cells in which insulin expression is inducible. Current progress in these studies will be reported.