Human artificial chromosome

HACs were first constructed de novo in 1997 by adding alpha-satellite DNA to telomeric and genomic DNA in human HT1080 cells. This resulted in an entirely new microchromosome that contained DNA of interest, as well as elements allowing it to be structurally and mitotically stable, such as telomeric and centromeric sequences.{{cite journal | vauthors = Harrington JJ, Van Bokkelen G, Mays RW, Gustashaw K, Willard HF | title = Formation of de novo centromeres and construction of first-generation human artificial microchromosomes | journal = Nature Genetics | volume = 15 | issue = 4 | pages = 345–55 | date = April 1997 | pmid = 9090378 | doi = 10.1038/ng0497-345 | s2cid = 9150827 }} Due to the difficulty of de novo HAC formation, this method has largely been abandoned.

There are currently two accepted models for the creation of human artificial chromosome vectors. The first is to create a small minichromosome by altering a natural human chromosome. This is accomplished by truncating the natural chromosome, followed by the introduction of unique genetic material via the Cre-Lox system of recombination. The second method involves the literal creation of a novel chromosome de novo.{{cite journal | vauthors = Kakeda M, Hiratsuka M, Nagata K, Kuroiwa Y, Kakitani M, Katoh M, Oshimura M, Tomizuka K | title = Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts | journal = Gene Therapy | volume = 12 | issue = 10 | pages = 852–6 | date = May 2005 | pmid = 15750614 | doi = 10.1038/sj.gt.3302483 | doi-access = free }} Progress regarding de novo HAC formation has been limited, as many large genomic fragments will not successfully integrate into de novo vectors. Another factor limiting de novo vector formation is limited knowledge of what elements are required for construction, specifically centromeric sequences. Challenges involving centromeric sequences are being overcome.{{Cite journal|last1=Logsdon|first1=Glennis A.|last2=Gambogi|first2=Craig W.|last3=Liskovykh|first3=Mikhail A.|last4=Barrey|first4=Evelyne J.|last5=Larionov|first5=Vladimir|last6=Miga|first6=Karen H.|author-link6=Karen Miga|last7=Heun|first7=Patrick|last8=Black|first8=Ben E.|date=2019-07-25|title=Human Artificial Chromosomes that Bypass Centromeric DNA|journal=Cell|volume=178|issue=3|pages=624–639.e19|doi=10.1016/j.cell.2019.06.006|issn=0092-8674|pmc=6657561|pmid=31348889}}

A 2009 study has shown additional benefits of HACs, namely their ability to stably contain extremely large genomic fragments. Researchers incorporated the 2.4{{nbsp}}Mb dystrophin gene, in which a mutation is a key causal element of Duchenne muscular dystrophy. The resulting HAC was mitotically stable, and correctly expressed dystrophin in chimeric mice. Previous attempts at correctly expressing dystrophin have failed. Due to its large size, it has never before been successfully integrated into a vector.{{cite journal | vauthors = Hoshiya H, Kazuki Y, Abe S, Takiguchi M, Kajitani N, Watanabe Y, Yoshino T, Shirayoshi Y, Higaki K, Messina G, Cossu G, Oshimura M | title = A highly stable and nonintegrated human artificial chromosome (HAC) containing the 2.4 Mb entire human dystrophin gene | journal = Molecular Therapy | volume = 17 | issue = 2 | pages = 309–17 | date = February 2009 | pmid = 19034264 | pmc = 2835068 | doi = 10.1038/mt.2008.253 | url = }}

In 2010, a refined human artificial chromosome called 21HAC was reported. 21HAC is based on a stripped copy of human chromosome 21, producing a chromosome 5{{nbsp}}Mb in length. Truncation of chromosome 21 resulted in a human artificial chromosome that was mitotically stable. 21HAC was also able to be transferred into cells from a variety of species (mice, chickens, humans). Using 21HAC, researchers were able to insert a herpes simplex virus thymidine kinase coding gene into tumor cells. This "suicide gene" is required to activate many antiviral medications. These targeted tumor cells were successfully, and selectively, terminated by the antiviral drug ganciclovir in a population including healthy cells. This research opens a variety of opportunities for using HACs in gene therapy.{{cite journal | vauthors = Kazuki Y, Hoshiya H, Takiguchi M, Abe S, Iida Y, Osaki M, Katoh M, Hiratsuka M, Shirayoshi Y, Hiramatsu K, Ueno E, Kajitani N, Yoshino T, Kazuki K, Ishihara C, Takehara S, Tsuji S, Ejima F, Toyoda A, Sakaki Y, Larionov V, Kouprina N, Oshimura M | title = Refined human artificial chromosome vectors for gene therapy and animal transgenesis | journal = Gene Therapy | volume = 18 | issue = 4 | pages = 384–93 | date = April 2011 | pmid = 21085194 | pmc = 3125098 | doi = 10.1038/gt.2010.147 }}

In 2011, researchers formed a human artificial chromosome by truncating chromosome 14. Genetic material was then introduced using the Cre-Lox recombination system. This particular study focused on changes in expression levels by leaving portions of the existing genomic DNA. By leaving existing telomeric and sub-telomeric sequences, researchers were able to amplify expression levels of genes coding for erythropoietin production over 1000-fold. This work also has large gene therapy implications, as erythropoietin controls red blood cell formation.{{cite journal | vauthors = Kakeda M, Nagata K, Osawa K, Matsuno H, Hiratsuka M, Sano A, Okazaki A, Shitara S, Nishikawa S, Masuya A, Hata T, Wako S, Osaki M, Kazuki Y, Oshimura M, Tomizuka K | title = A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells | journal = Biochemical and Biophysical Research Communications | volume = 415 | issue = 3 | pages = 439–44 | date = November 2011 | pmid = 22051050 | doi = 10.1016/j.bbrc.2011.10.088 }}

HACs have been used to create transgenic animals for use as animal models of human disease and for production of therapeutic products.

• Plasmid
• Cosmid
• Fosmid

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{{Nucleic acids}}

Category:Molecular biology