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Human Artificial Chromosomes Gene Therapy (2009) 16, 1180–1188 & 2009 Macmillan Publishers Limited All rights reserved 0969-7128/09 $32.00 www.nature.com/gt REVIEW Progress and prospects: human artificial chromosomes S Macnab1,2 and A Whitehouse1,2 1Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK and 2Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Artificial chromosomes (ACs) are highly promising vectors technically challenging to construct and diffcult to deliver to for use in gene therapy applications. They are able to maintain target cells. This review focuses on the current progress in the expression of genomic-sized exogenous transgenes within field of ACs and discusses the recent advances in purification, target cells, without integrating into the host genome. Although construction, delivery and potential new molecular therapies. these vectors have huge potential and benefits when compared Gene Therapy (2009) 16, 1180–1188; doi:10.1038/gt.2009.102; against normal expression constructs, they are highly complex, published online 27 August 2009 In brief Progress Propects Advances in the purification of YACs. Improvements to HAC and YAC purification proto- A new transposon-based technique for the generation cols. of novel virus-based BACs. Development of new or improved HAC and YAC Amplicon virus-like particles present efficient deliv- transduction methods for in vivo delivery will be ery mechanisms for ACs. developed. Hybrid AC technologies take advantage of the best Removal of contaminating virus from high titre traits of different vectors. amplicon delivery systems. AYAC-based method for genomic vector construction Use of genomic transcripts is viable and will reduce using TAR cloning. the need for cDNA transgenes. A novel method for BAC transgene molecular Further developments combining artificial chromo- tagging. somes and stem-cell therapeutics. BAC construct size may not affect in vivo vector The use of artificial chromosomes to treat a wide delivery. range of genetic diseases. Advances using ACs to deliver a wide variety of transgenes including CFTR, FRDA and dystrophin. ACs and stem-cell-based therapies. Keywords: HACs; BACs; YACs; amplicons; genomic transgenes; stem cells Introduction headings of human artificial chromosomes (HACs), bacterial artificial chromosomes (BACs), yeast artificial Artificial chromosomes (ACs) are promising tools for chromosomes (YACs) and P1-derived artificial chromo- gene therapy applications and possess many advantages somes (PACs).1,2 over current vector systems. Similar to endogenous The major advantages of ACs are their potential to chromosomes, ACs can replicate and segregate autono- overcome problems in gene therapy protocols such mously without integration into the host chromosome. as immunogenicity, insertional mutagenesis, oncogene There are a number of artificial chromosomal systems activation or limitations in capacity for transgene currently under development, which fall under the broad expression. Whether the AC is an HAC, BAC, YAC or PAC, the basic requirement for an AC remains the same; the AC needs to be independently transferred to progeny Correspondence: Dr A Whitehouse, Institute of Molecular and cells in a dividing cell population, although continuing Cellular Biology, Faculty of Biological Sciences, University of Leeds, to maintain spatial and temporal levels of long-term Leeds, LS2 9JT, UK. 3 E-mail: [email protected] transgene expression in specific cell types. In addition, Received 30 January 2009; revised 25 July 2009; accepted 28 July the AC should present no risk of cellular transformation 2009; published online 27 August 2009 or adverse immune system stimulation. Human artificial chromosomes S Macnab and A Whitehouse 1181 ACs can broadly be classified into two groups: linear 250 kb in size. An advantage of this methodology is that ACs and circular ACs. If a linear form of AC is used, it it is quicker than the pulse-field gel electrophoresis requires a number of cis-acting elements for successful purification method. In addition, it does not require the maintenance in vivo: (i) specific origins of replication, to use of time consuming CsCL-EthBr gradients of other maintain faithful DNA replication; (ii) a centromere, large DNA purification protocols. However, this system used for maintaining stability during the cell cycle and allows for the production and purification of vectors mitotic segregation and (iii) DNA capping telomeres, to containing genomic regions that are potentially unstable maintain linearity.4 Linear ACs have a potential for in standard Escherichia coli vectors. This technique is, unlimited transgene capacity. therefore, important as it may be suitable for carrying out Circular ACs are vectors generally produced from investigations into the human genome that current BAC BACs or PACs and have reasonably large transgene library-based shotgun protocols cannot achieve, as it is capacities, typically 300 kb, but have a maximum well documented that long inverted repeats, AT-rich potential of 700 kb. Other advantages include ease of sequences, are more stable in yeast than in E. coli.1,7 production and purification.2,5 Circular ACs require an However, as these vectors are produced in an organism origin of replication and an episomal maintenance that has minimal methyltransferase activity, this may element or a method of chromosome integration. In ultimately have implications in transgene expression in addition, if they are to be delivered by an infectious human beings, which have highly methylated DNA. agent, the appropriate packaging signal needs to be included in the plasmid. These requirements are often fulfilled by the use of viral elements. A new transposon-based technique for the generation of novel virus-based BACs Advances in the purification of YACs BAC and PAC vectors allow for quick, high yield, vector production (up to 500 mg DNA from 1 l culture). ACs can Production and purification of large size vectors such as be produced from BACs and PACs by the inclusion of YACs and HACs are problematic, not only because of episomal maintenance elements, usually of viral origin.2 low copy number vectors, which require purification In addition, the development of BAC technology has from large volumes of culture, but also because of the allowed the modification of large viral genomes to physical properties of the vector. become a viable tool for generating self-packaging AC A number of different protocols have recently been delivery vectors. A recent advance in the field of BAC developed, which provide methodologies for producing generation has used a sequence-independent in vitro YAC DNA, at concentrations typically 5–10 ng mlÀ1, and transposon-based methodology.9 This technique allows also reduces the likelihood of YAC sheering.1,6 One the construction of viral BACs through the use of a Tn5 successful protocol involves separating purified yeast transposition system. In brief, the authors constructed a chromosomes (including the YAC) through pulse-field linear vector using a plasmid containing the necessary gel electrophoresis. The YAC band is then excised from mini-F sequence required for BAC propagation in the agarose matrix, turned vertically and embedded in a bacteria, the chloramphenicol resistance gene, a GFP high percentage, low-melting point agarose. A second fluorescent marker and two Tn5 transposase recognition electrophoresis step concentrates the YACs into a single sequences. The linear construct is then incubated with point. The YAC spot can then be melted releasing the wild-type viral genome in vitro allowing random concentrated intact YACs, which can then be used for transposition to occur in the presence of a transposase. micro-injection. This methodology has proved successful The constructs are then propagated and screened using for producing YACs suitable for the transgenesis conventional BAC techniques. In vitro transposition of mice.6 alleviates the need for prior knowledge of the viral However, pulse-field gel electrophoresis can be time sequences, or the cloning of viral fragments, which may consuming and, therefore, a second YAC purification not be feasible in fastidious large-genome viruses. method has been developed for the purification of Importantly, this technique could be used to discover circular YACs containing genomic transcripts. This earlier unidentified viruses, which could be subse- technique uses transformation-associated recombination quently used as gene therapy vectors or ACs. (TAR) cloning and exonuclease chromatography purifi- cation.7,8 TAR cloning is based on co-transduction into yeast spheroplasts of isolated genomic DNA along with a Amplicon virus-like particles present 0 0 vector construct that contains 5 and 3 homologous efficient delivery mechanisms for ACs sequences specific for a gene of interest. Recombination between the vector and the target DNA establishes a Although viral-based BACs have the potential to fulfil YAC. This methodology allows entire genes and large the aforementioned criteria of ACs, they still have chromosomal regions to be isolated from total genomic limitations associated with immunogenicity, total nucleic DNA by in vitro recombination in yeast without the need acid capacity and, similar to all large constructs, to construct YAC or BAC libraries. In particular, the transduction efficacy. These limitations have led to the authors have shown that this technique is able to production and continued development of amplicon produce circular
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