The research work in Dr Larin Monaco’s group at the Wellcome Trust Centre for Human Genetics, University of Oxford is focussed on the development of mammalian artificial chromosomes (MAC), including human artificial chromosomes (HACs) as novel gene expression vectors. We are interested in this approach as it offers significant advantages for studying the regulation and long term expression of genes in mammalian cells compared to existing systems. HAC vectors contain the essential elements for human chromosome function and stability including alpha satellite (alphoid) DNA, which is the major component of human centromeres and forms a functional de nova centromere in a human artificial chromosome. HAC therefore replicate and segregate as a normal chromosome avoiding the problems of integration into the host genome, and can accommodate entire genes including the regulatory elements, which should contribute to the development of long term gene expression. We generated HAC containing human chromosome 17 alphoid DNA and the entire human hypoxanthine phosphoribosyltranferase (HPRT) genomic locus that complemented the metabolic defect in HPRT deficient human cells. In a further comparative study we determined that efficient HAC formation is dependent on chromosome specific alphoid DNA templates. HAC vectors containing chromosome 17 alphoid DNA generated de novo HAC in human cells at a much higher frequency compare to similar vectors with chromosome Y alphoid DNA. This work was one of the first reports of successful gene transfer and complementation of a deficiency in human cells using a HAC vector, and demonstrates that chromosome 17 alphoid HAC are viable gene transfer vectors for studying gene expression of large genes in their genomic context.
Recently, we developed an alternative method for HAC formation in different cell lines, based on HSV-amplicon delivery. This technique consists in packaging the desired vector into Herpes Simplex-1 (HSV-1) capsids, via a helper virus free system. The viral particles (amplicons) are then used to infect the target cells. Because no self-replicating, wild type virus can be produced this technique is safe and effective. The high efficiency of the HSV-amplicon delivery allowed us to obtain HAC formation in several human cell types. These experiments also allowed us to identify elements of the host cell genetic background which are important for HAC formation and stability. The HSV-amplicon approach is currently being used to generate HAC in human stem cells (hES), and in induced pluripotent stem cells (iPS), in view of testing the potential of HAC as vector for gene therapy.
Due to the importance of murine models for the study of human genetic disease, it is essential to establish HAC in murine cells including embryonic stem cells. To this aim, HSV-HAC vectors based on human alpha satellite, and murine major and minor satellite are being use to transduct murine stem cells and primary fibroblasts.
HAC are a valuable tool in the analysis of complex chromatin structures such as the human centromere because of their small size and relative simplicity compared to normal human chromosomes. In another line of research, we are conducting a comprehensive study of the centromere and chromatin composition of HAC expressing human genes generated in human and murine cells.
Moralli D, Chan DY, Jefferson A, Volpi EV, Monaco ZL (2009). HAC stability in murine cells is influenced by nuclear localization and chromatin organization. BMC Cell Biol 10: 18.
Moralli D, Monaco ZL (2009). Simultaneous detection of FISH signals and bromo-deoxyuridine incorporation in fixed tissue cultured cells. PLoS ONE 4: e4483.
Moralli D, Simpson KM, Wade-Martins R, Monaco ZL (2006). A novel human artificial chromosome gene expression system using herpes simplex virus type 1 vectors. EMBO Rep 7: 911-8.
Monaco ZL, Moralli D (2006). Progress in artificial chromosome technology. Biochem Soc Trans 34: 324-7.
Grimes BR, Monaco ZL (2005). Artificial and engineered chromosomes: developments and prospects for gene therapy. Chromosoma 114: 230-41.
Wellcome Trust, Dystrophic Epidermolysis Bullosa Research Association UK
Chromosome Biology, Gene Therapy
Human and murine artificial chromosomes, human and murine stem cells, gene therapy, chromosome structure, chromosome segregation