New Vectors for Gene Delivery: Human and Mouse Artificial Chromosomes

Abstract

Human artificial chromosomes (HACs) have been generated mainly by either a ‘top‐down approach’ (engineered creation) or a ‘bottom‐up approach’ (de novo creation). HACs with acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. Mouse artificial chromosomes (MACs) with acceptor sites were also created from a native mouse chromosome. The microcell‐mediated chromosome transfer (MMCT) technique for manipulating HACs and MACs in donor cells in order to deliver them to recipient cells is required for each approach. This review describes the lessons learned and prospects identified from studies on the construction of HACs and MACs and their ability to drive exogenous gene expression in cultured cells and transgenic animals via MMCT. The recent emergence of stem cell‐based tissue engineering has opened up new avenues for gene and cell therapies, and possible applications for medical use of HACs and MACs are also proposed.

Key Concepts:

  • The microcell‐mediated chromosome transfer method can transfer an intact chromosome or chromosome fragment from donor cell to recipient cell.

  • Human artificial chromosome (HAC), which is an episomal vector derived from a native chromosome, has several advantages: it can contain the entire genome sequences of interest including its regulatory regions with long‐term stable maintenance and gene expression in human cells.

  • The de novo HAC (bottom‐up approach) has been developed with alphoid DNA in HT1080 cells under the condition with lower H3K9me3 methylated alphoid DNA concerning cell‐type‐specific barrier for de novo CENP‐A assembly.

  • The tet‐O HAC is a kind of de novo HAC with a conditional centromere which is constructed with alphoid DNA, and tetracycline operator sequence can be inactivated by expression of tet‐repressor fusion protein for loss of the tet‐O HAC.

  • Mouse artificial chromosome (MAC) is a useful tool for the animal transgenesis because it can be maintained stably in mouse cells and transchromosomic mice through germline transmission.

  • HAC/MAC have a loxP site and/or five recombination platforms to insert circular vectors like bacterial artificial chromosome or make a translocation of the targeting region from other chromosomes.

  • HACs could be used for the complete correction of iPS cells from a patient with Duchenne muscular dystrophy.

  • An HAC which contains Klf4, Sox2, Oct4, c‐Myc and siRNA expression against p53 could reprogramme mouse embryonic fibroblast to pluripotent stem cells.

  • Humanised model mice using human artificial chromosomes, which contain the human CYP3A cluster and the human immunoglobulins including whole 700kb or 1Mb genomic region, have been produced.

  • HACs/MACs might be used for a wide variety of purposes including medical and pharmaceutical applications and even for synthetic biology in sophisticated control.

Keywords: human artificial chromosome; mouse artificial chromosome; induced pluripotent stem cell; embryonic stem cell; microcell‐mediated chromosome transfer; gene therapy; Duchenne muscular dystrophy; humanised model mouse; xenobiotic metabolism

Figure 1.

Construction of mouse A9 hybrid cells carrying a single human chromosome by MMCT. The first step involves marking the human chromosome in the fibroblasts with a selection marker and fusing the fibroblasts with mouse A9 cells. The second step is the introduction of the marked human chromosome from the donor hybrid to the recipient A9 cells. The procedure can be divided into several parts: micronucleation of the donor hybrids by colcemid treatment, enucleation in the presence of cytochalasin B, purification of the microcells, fusion with the recipient A9 cells, drug selection of the microcell hybrids, identification of the transferred human chromosome by fluorescence in situ hybridisation and DNA analyses.

Figure 2.

Potential characteristics of HACs. (a) Size limits for gene delivery vectors. The maximum deliverable DNA size in each vector is described. HAC vectors as well as chromosomes can carry DNA fragments larger than 1 Mb size. The size limits depend on each vector. (b, c) Limitations and consequences of gene delivery with conventional vectors such as viruses or plasmids and with HACs.

Figure 3.

Two types of gene loading to HAC. (a) Construction of a HAC vector from human chromosome 21 using the top‐down approach. The 21HAC is equipped with a loxP site for loading the gene of interest. A site‐specific recombination event mediated by Cre recombinase is selected by reconstruction of the functional HPRT gene, which confers HAT resistance. (b) The gene of interest, isolated in a circular vector, is introduced into the HAC by site‐specific insertion. (c) A megabase size gene locus, which is above the capacity of circular cloning vectors, is introduced into the HAC by site‐specific reciprocal chromosome translocation.

Figure 4.

Examples of HAC/MAC using chromosome engineering techniques, which are based on chromosome transfer, have been applied for gene therapy, regenerative medicine, production of humanised model animals and production of therapeutic substances in TC livestock for industrial purposes. ES cells, embryonic stem cells.

Figure 5.

Future applications of HAC and MAC vectors.

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References

Basu J, Stromberg G, Compitello G, Willard HF and Van Bokkelen G (2005) Rapid creation of BAC‐based human artificial chromosome vectors by transposition with synthetic alpha‐satellite arrays. Nucleic Acids Research 33: 587–596.

Brown KE, Barnett MA, Burgtorf C et al. (1994) Dissecting the centromere of the human Y chromosome with cloned telomeric DNA. Human Molecular Genetics 3: 1227–1237.

Buerstedde JM and Takeda S (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67: 179–188.

Devoy A, Bunton‐Stasyshyn RK, Tybulewicz VL, Smith AJ and Fisher EM (2011) Genomically humanized mice: technologies and promises. Nature Reviews Genetics 13(1): 14–20.

Farr CJ, Stevanovic M, Thomson EJ, Goodfellow PN and Cooke HJ (1992) Telomere‐associated chromosome fragmentation: applications in genome manipulation and analysis. Nature Genetics 2: 275–282.

Harrington JJ, Van Bokkelen G, Mays RW, Gustashaw K and Willard HF (1997) Formation of de novo centromeres and construction of first‐generation human artificial microchromosomes. Nature Genetics 15: 345–355.

Heller R, Brown KE, Burgtorf C and Brown WR (1996) Mini‐chromosomes derived from the human Y chromosome by telomere directed chromosome breakage. Proceedings of the National Academy of Sciences of the USA 93: 7125–7130.

Hiratsuka M, Uno N, Ueda K et al. (2011) Integration‐free iPS cells engineered using human artificial chromosome vectors. PLos One 6(10): e25961.

Hoshiya H, Kazuki Y, Abe S et al. (2009) A highly stable and nonintegrated human artificial chromosome (HAC) containing the 2.4 Mb entire human dystrophin gene. Molecular Therapy 17: 309–317.

Iida Y, Kim JH, Kazuki Y et al. (2010) Human artificial chromosome with a conditional centromere for gene delivery and gene expression. DNA Research 17: 293–301.

Ikeno M, Grimes B, Okazaki T et al. (1998) Construction of YAC‐based mammalian artificial chromosomes. Nature Biotechnology 16: 431–439.

Kakeda M, Hiratsuka M, Nagata K et al. (2005) Human artificial chromosome (HAC) vector provides long‐term therapeutic transgene expression in normal human primary fibroblasts. Gene Therapy 12(10): 852–856.

Kanatsu‐Shinohara M, Inoue K, Lee J et al. (2004) Generation of pluripotent stem cells from neonatal mouse testis. Cell 119: 1001–1012.

Katoh M, Ayabe F, Norikane S et al. (2004) Construction of a novel human artificial chromosome vector for gene delivery. Biochemical and Biophysical Research Communications 321(2): 280–290.

Katoh M, Kazuki Y, Kazuki K et al. (2010) Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell‐mediated chromosome transfer. BMC Biotechnology 10: 37.

Katona RL, Sinkó I, Holló G et al. (2008) A combined artificial chromosome‐stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse. Cellular and Molecular Life Sciences 65(23): 3830–3838.

Kazuki Y and Oshimura M (2011) Human artificial chromosomes for gene delivery and the development of animal models. Molecular Therapy 19(9): 1591–1601.

Kazuki Y, Hiratsuka M, Takiguchi M et al. (2010) Complete genetic correction of ips cells from Duchenne muscular dystrophy. Molecular Therapy 18: 386–393.

Kazuki Y, Hoshiya H, Takiguchi M et al. (2011) Refined human artificial chromosome vectors for gene therapy and animal transgenesis. Gene Therapy 18: 384–393.

Kazuki Y, Kobayashi K, Aueviriyavit S et al. (2012) Trans‐chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans. Human Molecular Genetics (in press).

Kim JH, Kononenko A, Erliandri I et al. (2011) Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells. Proceedings of the National Academy of Sciences of the USA 108(50): 20048–20053.

Koenig M, Hoffman EP, Bertelson CJ et al. (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50: 509–517.

Kohn DB, Weinberg KI, Nolta JA et al. (1995) Engraftment of gene‐modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nature Medicine 1: 1017–1023.

Kotzamanis G, Cheung W, Abdulrazzak H et al. (2005) Construction of human artificial chromosome vectors by recombineering. Gene 351: 29–38.

Kouprina N, Earnshaw WC, Masumoto H and Larionov V (2012) A new generation of human artificial chromosomes for functional genomics and gene therapy. Cellular and Molecular Life Sciences (Epub ahead of print).

Kouprina N, Ebersole T, Koriabine M et al. (2003) Cloning of human centromeres by transformation‐associated recombination in yeast and generation of functional human artificial chromosomes. Nucleic Acids Research 31: 922–934.

Kuroiwa Y, Kasinathan P, Sathiyaseelan T et al. (2009) Antigen‐specific human polyclonal antibodies from hyperimmunized cattle. Nature Biotechnology 27: 173–181.

Kurosaki H, Hiratsuka M, Imaoka N et al. (2011) Integration‐free and stable expression of FVIII using a human artificial chromosome. Journal of Human Genetics 56(10): 727–733.

Lindenbaum M, Perkins Ed, Csonka E et al. (2004) A mammalian artificial chromosome engineering system applicable to biopharmaceutical protein production, transgenesis and gene‐based cell therapy. Nucleic Acids Research 32(21): e172.

Mandegar MA, Moralli D, Khoja S et al. (2011) Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells. Human Molecular Genetics 20(15): 2905–2913.

Meaburn KJ, Parris CN and Bridger JM (2005) The manipulation of chromosomes by mankind: the uses of microcell‐mediated chromosome transfer. Chromosoma 114(4): 263–274.

Monaco ZL and Moralli D (2006) Progress in artificial chromosome technology. Biochemical Society Transactions 34: 324–327.

Moralli D, Simpson KM, Wade‐Martins R and Monaco ZL (2006) A novel human artificial chromosome gene expression system using herpes simplex virus type 1 vectors. EMBO Reports 7: 911–918.

Nakano M, Cardinale S, Noskov VN et al. (2008) Inactivation of a human kinetochore by specific targeting of chromatin modifiers. Devolopmental Cell 14: 507–522.

Narayanan K and Warburton PE (2003) DNA modification and functional delivery into human cells using E. coli DH10B. Nucleic Acids Research 31: e51.

Odom GL, Gregorevic P and Chamberlain JS (2007) Viral‐mediated gene therapy for the muscular dystrophies: successes, limitations and recent advances. Biochimica et Biophysica Acta 1772: 243–262.

Ohzeki J, Bergmann JH, Kouprina N et al. (2012) Breaking the HAC Barrier: histone H3K9 acetyl/methyl balance regulates CENP‐A assembly. EMBO Journal 31(10): 2391–2402.

Ren X, Katoh M, Hoshiya H et al. (2005) A novel human artificial chromosome vector provides effective cell lineage‐specific transgene expression in human mesenchymal stem cells. Stem Cells 23: 1608–1616.

Sampaolesi M, Blot S, D'Antona G et al. (2006) Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 444: 574–579.

Shinohara T, Tomizuka K, Miyabara S et al. (2001) Mice containing a human chromosome 21 model behavioral impairment and cardiac anomalies of Down's syndrome. Human Molecular Genetics 10: 1163–1175.

Suzuki N, Nishii K, Okazaki T and Ikeno M (2006) Human artificial chromosomes constructed using the bottom‐up strategy are stably maintained in mitosis and efficiently transmissible to progeny mice. Journal of Biological Chemistry 281: 26615–26623.

Takahashi K and Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676.

Takiguchi M, Kazuki Y, Hiramatsu K et al. (2012) A novel and stable mouse artificial chromosome vector. Synthetic Biology (in press).

Tedesco FS, Gerli MF, Perani L et al. (2012) Transplantation of genetically corrected human iPSC‐derived progenitors in mice with limb‐girdle muscular dystrophy. Science Translational Medicine 4(140): 140ra89.

Tedesco FS, Hoshiya H, D'Antona G et al. (2011) Stem cell‐mediated transfer of a human artificial chromosome ameliorates muscular dystrophy. Science ranslational Medicine 3(96): 96ra78.

Tomizuka K, Yoshida H, Uejima H et al. (1997) Functional expression and germline transmission of a human chromosome fragment in chimaeric mice. Nature Genetics 16: 133–143.

Voet T, Schoenmakers E, Carpentier S, Labaere C and Marynen P (2003) Controlled transgene dosage and PAC‐mediated transgenesis in mice using a chromosomal vector. Genomics 82(6): 596–605.

Yamaguchi S, Kazuki Y, Nakayama Y et al. (2011) A method for producing transgenic cells using a multi‐integrase system on a human artificial chromosome vector. PLoS One 6: e17267.

Further Reading

Basu J and Willard HF (2006) Human artificial chromosomes: potential applications and clinical considerations. Pediatric Clinics of North America 53: 843–853.

Grimes BR and Monaco ZL (2005) Artificial and engineered chromosomes: developments and prospects for gene therapy. Chromosoma 114(4): 230–241.

Kazuki Y, Schulz TC, Shinohara T et al. (2003) A new mouse model for Down syndrome. Journal of Neural Transmission Supplementum (67): 1–20.

Okita K and Yamanaka S (2011) Induced pluripotent stem cells: opportunities and challenges. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 366(1575): 2198–2207.

Ringertz RN and Savage ER (1977) Cell Hybrids. San Diego: Academic Press Inc., ISBN‐10: 0125891504, ISBN‐13: 978‐0125891509.

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Oshimura, Mitsuo, Kazuki, Yasuhiro, Iida, Yuichi, and Uno, Narumi(Feb 2013) New Vectors for Gene Delivery: Human and Mouse Artificial Chromosomes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0024474]