Human Artificial Chromosome (HAC) Vector with a Conditional Centromere for Correction of Genetic Deficiencies in Human Cells

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2011 Nov 30

PMID 22123967

Citations 49

Authors

Jung-Hyun Kim

Artem Kononenko

Indri Erliandri

Tae-Aug Kim

Megumi Nakano

Yuichi Iida

J Carl Barrett

Mitsuo Oshimura

Hiroshi Masumoto

William C Earnshaw

Vladimir Larionov

Natalay Kouprina

Affiliations

Soon will be listed here.

Abstract

Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. HACs avoid the limited cloning capacity, lack of copy number control, and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression of NBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications.

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References

Kouprina N, Larionov V . Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat Protoc. 2008; 3(3):371-7. DOI: 10.1038/nprot.2008.5. View

Iida Y, Kim J, Kazuki Y, Hoshiya H, Takiguchi M, Hayashi M . Human artificial chromosome with a conditional centromere for gene delivery and gene expression. DNA Res. 2010; 17(5):293-301. PMC: 2955713. DOI: 10.1093/dnares/dsq020. View

Monaco Z, Moralli D . Progress in artificial chromosome technology. Biochem Soc Trans. 2006; 34(Pt 2):324-7. DOI: 10.1042/BST20060324. View

Noon A, Shibata A, Rief N, Lobrich M, Stewart G, Jeggo P . 53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair. Nat Cell Biol. 2010; 12(2):177-84. DOI: 10.1038/ncb2017. View

Maier P, von Kalle C, Laufs S . Retroviral vectors for gene therapy. Future Microbiol. 2010; 5(10):1507-23. DOI: 10.2217/fmb.10.100. View

Kazuki Y, Hoshiya H, Takiguchi M, Abe S, Iida Y, Osaki M . Refined human artificial chromosome vectors for gene therapy and animal transgenesis. Gene Ther. 2010; 18(4):384-93. PMC: 3125098. DOI: 10.1038/gt.2010.147. View

Tauchi H, Matsuura S, Kobayashi J, Sakamoto S, Komatsu K . Nijmegen breakage syndrome gene, NBS1, and molecular links to factors for genome stability. Oncogene. 2002; 21(58):8967-80. DOI: 10.1038/sj.onc.1206136. View

Kazuki Y, Hiratsuka M, Takiguchi M, Osaki M, Kajitani N, Hoshiya H . Complete genetic correction of ips cells from Duchenne muscular dystrophy. Mol Ther. 2009; 18(2):386-93. PMC: 2839293. DOI: 10.1038/mt.2009.274. View

Grimes B, Larin Monaco Z . Artificial and engineered chromosomes: developments and prospects for gene therapy. Chromosoma. 2005; 114(4):230-41. DOI: 10.1007/s00412-005-0017-5. View

10.

Koochekpour S, Jeffers M, Wang P, Gong C, Taylor G, Roessler L . The von Hippel-Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells. Mol Cell Biol. 1999; 19(9):5902-12. PMC: 84441. DOI: 10.1128/MCB.19.9.5902. View

11.

Shen M, Mee P, Nichols J, Yang J, Brook F, Gardner R . A structurally defined mini-chromosome vector for the mouse germ line. Curr Biol. 2000; 10(1):31-4. DOI: 10.1016/s0960-9822(99)00261-4. View

12.

Maxwell P, Wiesener M, Chang G, Clifford S, Vaux E, Cockman M . The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999; 399(6733):271-5. DOI: 10.1038/20459. View

13.

Matrai J, Chuah M, VandenDriessche T . Recent advances in lentiviral vector development and applications. Mol Ther. 2010; 18(3):477-90. PMC: 2839421. DOI: 10.1038/mt.2009.319. View

14.

Mills W, Critcher R, Lee C, Farr C . Generation of an approximately 2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40. Hum Mol Genet. 1999; 8(5):751-61. DOI: 10.1093/hmg/8.5.751. View

15.

Li C, Park J, Simonaro C, He X, Gordon R, Ehleiter D . Insertional mutagenesis of the mouse acid ceramidase gene leads to early embryonic lethality in homozygotes and progressive lipid storage disease in heterozygotes. Genomics. 2002; 79(2):218-24. DOI: 10.1006/geno.2002.6686. View

16.

Takahashi K, Yamanaka S . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-76. DOI: 10.1016/j.cell.2006.07.024. View

17.

Ebersole T, Okamoto Y, Noskov V, Kouprina N, Kim J, Leem S . Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation. Nucleic Acids Res. 2005; 33(15):e130. PMC: 1197135. DOI: 10.1093/nar/gni129. View

18.

Leen Lam A, Boivin C, Bonney C, Rudd M, Sullivan B . Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA. Proc Natl Acad Sci U S A. 2006; 103(11):4186-91. PMC: 1449668. DOI: 10.1073/pnas.0507947103. View

19.

Breman A, Steiner C, Slee R, Grimes B . Input DNA ratio determines copy number of the 33 kb Factor IX gene on de novo human artificial chromosomes. Mol Ther. 2007; 16(2):315-23. DOI: 10.1038/sj.mt.6300361. View

20.

Spence J, Critcher R, Ebersole T, Valdivia M, Earnshaw W, Fukagawa T . Co-localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X alpha-satellite array. EMBO J. 2002; 21(19):5269-80. PMC: 129033. DOI: 10.1093/emboj/cdf511. View