Gene therapy is a technique for correcting defective genes responsible for disease development. In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal" disease-causing gene. Researchers are interested to find appropriate vectors to cure genetic diseases in a predictive and safe manner.
Artificial human chromosomes, bearing all the elements of a natural chromosome (telomeres, origins of replication, a centromere single copy and repetitive sequences) were considered as new vectors that can insure stability, copy number control and safely vectors without unpredicted insertion events in the human genome. Human artificial chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome.
Some other researchers (1) constructed HACs carrying a 180 kb genome segment encoding the human GCH1 gene and its control region from the bacterial artificial chromosome (BAC) with the GCH1 segment by co-transfection with the alpha-satellite DNA-containing BAC to a human fibroblast cell line. The GCH1 activities of the HAC-carrying human fibroblast cell lines were elevated but still highly sensitive to IFN-gamma induction, mimicking the response of the gene expression from the authentic chromosomal genes. These HACs will provide a useful system for analysis of the complex regulatory circuit of the GCH1 gene in vivo and also function as a tool for gene delivery in animal models or in therapeutic trials.
Mammalian artificial chromosomes (ACEs) transferred to autologous adult stem cells (SCs) provide a novel strategy for the ex vivo gene therapy of a variety of clinical indications. Unlike retroviral vectors, ACEs are stably maintained, autonomous, and nonintegrating. In their report another research group (2) assessed the delivery efficiency of ACEs and evaluated the subsequent differentiation potential of ACE-transfected bone marrow-derived human mesenchymal stem cells (hMSCs). It was demonstrated that the ACEs were stably maintained as single chromosomes and expressed the RFP transgenes in both differentiated cultures.
Another group (3) studied the introduction of the HAC vector, which is reduced in size and devoid of most expressed genes, into normal primary human fibroblasts (hPFs) with microcell-mediated chromosome transfer (MMCT). They demonstrated the generation of cytogenetically normal hPFs harboring the structurally defined and extra HAC vector. This introduced HAC vector was retained stably in hPFs without translocation of the HAC on host chromosomes. They also achieved the long-term production of human erythropoietin for at least 12 weeks in them.
Other research groups (4-8, 12) have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. They have utilized this vector system to rapidly design, construct and validate multiple de novo HACs containing large (100-200 kb) genomic loci including therapeutically significant genes for human growth hormone (HGH), polycystic kidney disease (PKD1) and beta-globin. They have observed sustained beta-globin gene expression from HACs incorporating the entire 200 kb beta-globin genomic locus for over 90 days in the absence of selection (6). Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus.
Another group (9) demonstrated the feasibility of 21deltaqHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. They first introduced a 21deltaqHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21deltaqHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21deltaqHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21deltaqHAC could still be detected at two weeks post-transplantation in immunodeficient mice.
In this study, it was tried 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. Were 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.(10)
Aneuploidies are common chromosomal defects that result in growth and developmental deficits and high levels of lethality in humans. To gain insight into the biology of aneuploidies, were manipulated mouse embryonic stem cells and generated a trans-species aneuploid mouse line that stably transmits a freely segregating, almost complete human chromosome 21 (Hsa21). This transchromosomic mouse line, Tc1, is a model of trisomy 21, which manifests as Down syndrome (DS) in humans phenotypic alterations. Transchromosomic mouse lines such as Tc1 may represent useful genetic tools for dissecting other human aneuploidies (11)