Research Area:	Genetics and Genomics
Technology Exchange:	Chromosome mapping, ES cell / homologous recombination, Gene therapy, Immunohistochemistry and In situ hybridisation
Keywords:	Human Artificial Chromosomes, Gene therapy, Gene expression, Chromosome Structure, Centromere structure and Chromosome segregation

The research work in Dr Larin Monaco’s group is focussed on the development of mammalian artificial chromosomes (MACs), including human artificial chromosomes (HACs) as novel gene expression vectors.
HAC vectors contain the essential elements for human chromosome function and stability including alpha satellite (alphoid) DNA. HACs therefore replicate and segregate as a normal chromosome avoiding the problems of integration into the host genome, and can accomodate entire genes including the regulatory elements which should contribute to the development of long term gene expression.
Our work is one of the first reports of successful gene transfer and complementation of a deficiency in human cells using a HAC vector. HACs now offer the possibility to study human gene expression in a variety of cell types from different tissues and to complement gene deficiencies caused by human inherited diseases as a first step towards gene therapy. We aim to develop HAC expression systems that are generally applicable for studying a wide variety of genes. One gene of interest is human type VII collagen (COL7A1) which is mutated in dystrophic epidermolysis bullosa (DEB), a severe skin blistering disorder. We propose to complement the COL7A1 deficiency with HACs in cultured keratinocyte cells derived from DEB patients. Complementary studies in murine cells are also important to determine if large human transgenes can be expressed appropriately from an artificial chromosome especially for murine models of human genetic diseases. We are currently determining the components required for establishing stable artificial chromosomes in murine cells.

There are no collaborations listed for this principal investigator.

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 pp. 18. Read abstract | Read more

Human artificial chromosomes (HAC) are small functional extrachromosomal elements, which segregate correctly during each cell division. In human cells, they are mitotically stable, however when the HAC are transferred to murine cells they show an increased and variable rate of loss. In some cell lines the HAC are lost over a short period of time, while in others the HAC become stable without acquiring murine DNA. Hide abstract

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 (9), pp. 911-918. Read abstract | Read more

Human artificial chromosome (HAC) vectors are an important gene transfer system for expression and complementation studies. We describe a significant advance in HAC technology using infectious herpes simplex virus type 1 (HSV-1) amplicon vectors for delivery. This highly efficient method has allowed gene-expressing HACs to be established in glioma-, kidney- and lung-derived cells. We also developed an HSV-1 hypoxanthine phosphoribosyltransferase (HPRT) HAC vector, which generated functional HPRT-expressing HACs that complemented the genetic deficiency in human cells. The transduction efficiency of the HSV-1 HAC amplicons is several orders of magnitude higher than lipofection-mediated delivery. Studies on HAC stability between cell types showed important differences that have implications for HAC development and gene expression in human cells. This is the first report of establishing gene-expressing HACs in human cells by using an efficient, high-capacity viral vector and by identifying factors that are involved in cell-type-specific HAC instability. The work is a significant advance for HAC technology and the development of HAC gene expression systems in human cells. Hide abstract

Monaco ZL, Moralli D. 2006. Progress in artificial chromosome technology. Biochem Soc Trans, 34 (Pt 2), pp. 324-327. Read abstract | Read more

Artificial chromosomes is an exciting technology which has developed rapidly since the late 1990s. HACs (human artificial chromosomes) are autonomous molecules that can function and segregate as normal chromosomes in human cells. The advantages of an artificial-chromosome-based system are 2-fold. First, HACs are an excellent research tool for investigating the requirements for normal chromosome structure and function during the cell cycle. They are important in defining the sequence requirements of functional chromosomes, and investigating the organization and composition of the chromatin. Secondly, HACs are useful gene-transfer vectors for expression studies in mammalian cells, with the capacity to incorporate large DNA segments encompassing genes and their regulatory elements. As episomes, they are stably maintained, leading to more reliable and prolonged transgene expression. HACs offer the possibility of long-term gene expression in human cells and the development of future somatic gene therapy. Hide abstract

Grimes BR, Monaco ZL. 2005. Artificial and engineered chromosomes: developments and prospects for gene therapy. Chromosoma, 114 (4), pp. 230-241. Read abstract | Read more

At the gene therapy session of the ICCXV Chromosome Conference (2004), recent advances in the construction of engineered chromosomes and de novo human artificial chromosomes were presented. The long-term aims of these studies are to develop vectors as tools for studying genome and chromosome function and for delivering genes into cells for therapeutic applications. There are two primary advantages of chromosome-based vector systems over most conventional vectors for gene delivery. First, the transferred DNA can be stably maintained without the risks associated with insertion, and second, large DNA segments encompassing genes and their regulatory elements can be introduced, leading to more reliable transgene expression. There is clearly a need for safe and effective gene transfer vectors to correct genetic defects. Among the topics discussed at the gene therapy session and the main focus of this review are requirements for de novo human artificial chromosome formation, assembly of chromatin on de novo human artificial chromosomes, advances in vector construction, and chromosome transfer to cells and animals. Hide abstract

Alazami AM, Mejía JE, Monaco ZL. 2004. Human artificial chromosomes containing chromosome 17 alphoid DNA maintain an active centromere in murine cells but are not stable. Genomics, 83 (5), pp. 844-851. Read abstract | Read more

Human artificial chromosomes (HACs) are autonomous molecules that can function and segregate as normal chromosomes in human cells. De novo HACs have successfully been used as gene expression vectors to complement genetic deficiencies in human cultured cells. HACs now offer the possibility of studying the regulation and expression of large genes in a variety of cell types from different tissues and correcting gene deficiencies caused by human inherited diseases. Complementary gene expression studies in mice, especially in mouse models of human genetic diseases, are also important in determining if large human transgenes can be expressed appropriately from artificial chromosomes. Toward this aim we are establishing artificial chromosomes in murine cells as novel gene expression vectors. Initially we transferred HAC vectors into murine cells, but were unable to generate de novo HACs at a reasonable frequency. We then transferred HACs previously established in human HT1080 cells to three different murine cell types by microcell fusion, followed by positive selection. We observed that the HACs in murine cells bound centromere protein C (CENP-C), a marker of active centromeres, and were detected under selection but rapidly lost when selection was removed. These results suggest that the HACs maintain at least a partially functional centromere complex in murine cells, but other factors are required for stability and segregation. Artificial chromosomes containing mouse centromeric sequences may be required for better stability and maintenance in murine cells. Hide abstract

Larin Z, Mejía JE. 2002. Advances in human artificial chromosome technology. Trends Genet, 18 (6), pp. 313-319. Read abstract | Read more

Human artificial chromosome (HAC) technology has developed rapidly over the past four years. Recent reports show that HACs are useful gene transfer vectors in expression studies and important tools for determining human chromosome function. HACs have been used to complement gene deficiencies in human cultured cells by transfer of large genomic loci also containing the regulatory elements for appropriate expression. And, they now offer the possibility to express large human transgenes in animals, especially in mouse models of human genetic diseases. Hide abstract

Mejía JE, Alazami A, Willmott A, Marschall P, Levy E, Earnshaw WC, Larin Z. 2002. Efficiency of de novo centromere formation in human artificial chromosomes. Genomics, 79 (3), pp. 297-304. Read abstract | Read more

In a comparative study, we show that human artificial chromosome (HAC) vectors based on alpha-satellite (alphoid) DNA from chromosome 17 but not the Y chromosome regularly form HACs in HT1080 human cells. We constructed four structurally similar HAC vectors, two with chromosome 17 or Y alphoid DNA (17alpha, Yalpha) and two with 17alpha or Yalpha and the hypoxanthine guanine phosphoribosyltransferase locus (HPRT1). The 17alpha HAC vectors generated artificial minichromosomes in 32-79% of the HT1080 clones screened, compared with only approximately 4% for the Yalpha HAC vectors, indicating that Yalpha is inefficient at forming a de novo centromere. The 17alpha HAC vectors produced megabase-sized, circular HACs containing multiple copies of alphoid fragments (60-250 kb) interspersed with either vector or HPRT1 DNA. The 17alpha-HPRT1 HACs were less stable than those with 17alpha only, and these results may influence the design of new HAC gene transfer vectors. Hide abstract

Mejía JE, Willmott A, Levy E, Earnshaw WC, Larin Z. 2001. Functional complementation of a genetic deficiency with human artificial chromosomes. Am J Hum Genet, 69 (2), pp. 315-326. Read abstract | Read more

We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alternating alphoid and nonalphoid DNA segments derived only from the input DNA (within the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitotic stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of this human-gene-transfer technology. Hide abstract