Human Artificial Chromosomes

Home , Cloning vector, In vitro recombination

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 purification.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 YACs up to 120 and 150 kb in size, systems. This is a unique way of creating a gene delivery from 1.5 l cultures, producing yields in the region of 5– tool that mimics the viral vector’s ability for genome 10 mg. In addition, the authors hypothesise that this delivery and transgene expression, although minimising protocol can be used to produce larger YACs, although the exposure to harmful viral genes and increasing the TAR cloning may be limited to producing YACs of up to packaging potential of the vector.

Gene Therapy Human artificial chromosomes S Macnab and A Whitehouse 1182 Amplicons can be defined as plasmid-based vectors ment. Recent work by Grant et al.13 has produced HSV-1 containing minimal viral coding sequences, which are packaging cell lines, which use replication defective able to be maintained in a dividing cell population, as a HSVDICP4DICP27 vectors (JDTOZHE). ICP4 and ICP27 non-integrated episome. In addition, amplicon plasmids are genes essential in activating immediate early gene can be packaged into infectious virions, to form amplicon transcription of the viral lytic cascade. This work is virus-like particles (ampVLPs). This is achieved by important because it highlights the need to completely production of viral structural proteins, usually generated understand the kinetics of immediate early virus gene by some form of replication-deficient helper virus. To transcription when producing packaging cell lines. date, a number of amplicon systems have been produced Production of an ICP4 and ICP27 supplemented based on the following viruses, particularly members of JDTOZHE vero cell lines resulted in a threefold drop in the herpesvirus family. These include Herpes simplex recombinant amplicon titre, when compared against virus-1, Pseudorabies, Epstein–Barr virus and human wild-type production. Insights into the unpredicted drop cytomegalovirus.2,10 in titre arose when complementation studies revealed Recent advances in this field include the development that supplemented ICP4 was delayed in expression by of a recombinant g-2 herpesvirus saimiri (HVS) amplicon 8–10 h after vector transduction. This issue was ad- system, which has the potential to deliver upwards of dressed by using the ICP0 promoter, which resulted in 50–150 kb heterologous DNA.10 HVS has potential to be higher induction levels, and faster kinetics of ICP4, as used in a range of protocols, as it can infect a wide well as an additional 10-fold increase in vector yield.13 variety of cells including colorectal, breast, lung, liver, kidney and a variety of white blood cells, whilst being episomally maintained in a dividing cell population in Hybrid AC technologies take advantage 11,12 both in vitro and in vivo studies. Important in vivo of the best traits of different vectors studies regarding the ability of HVS to deliver and express transgenes include the expression of bovine The development and analysis of a wide array of gene growth hormone in New World primates, as well as therapy vectors has revealed that all vectors have their nude mouse tumour xenograft studies and intraperito- associated pros and cons (Table 1). Therefore, it seems neal and intravenous HVS injection in mice. These unlikely that there will be a single ‘perfect’ vector. As a studies determined systemic spread and persistence of consequence, work is ongoing to harness the best aspects the viral genome. The results thus far have shown that of a multitude of vectors. Work by Moralli et al.14 HVS is able to be delivered, maintained and express provides an example of combining the delivery efficacy exogenous transgenes in the host after infection.12 of ampVLPs, though harnessing the natural maintenance However, the HVS amplicon system currently has wild- mechanisms of HACs.14 The authors constructed a HAC type virus contamination, an issue that is faced by all capable of being delivered by HSV-based ampVLPs amplicon systems in their infancy, this will need to be (Figure 1a). Infectious delivery was achieved in both addressed before it can be used in gene therapy protocols. primary and cultured human cells including fibrosarco- Other amplicon research has focused on reducing ma, glioma, kidney and lung. Infectious delivery resulted wild-type virus contamination of ampVLP stocks by in the HAC providing GFP and hypoxanthinephosphor- producing helper packaging cell lines, which supply all iboysltransferase transgene expression. Importantly, the lytic cascade and necessary structural genes in trans. this technique is able to deliver the HAC to the cells The Herpes simplex virus-based amplicon systems are at of interest, at much higher efficacies (up to 104 times the forefront of packaging cell line and amplicon greater, m.o.i. 1–10) than earlier HAC lipofectamine technology, elucidating the requirements for minimal transfections.2 In addition, transgene expression was wild-type contamination and amplicon vector develop- shown to be maintained and unchanged for a period of 3

Table 1 Details of the advantages and disadvantages of different AC technologies

Type Transgene capacity Advantages Disadvantages

HAC Unlimited Large transgene capacity Fragile construct Naturally segregates in human cells Difficult to purify Non-immunogenic Low transduction efficacy Non-integrating YAC Up to 2.5 Mb Large transgene capacity Fragile construct Ease of production and modification Low transduction efficacy BAC/PAC Up to 300 kb Can be infectiously delivered (if viral based) Medium transgene capacity Ease of purification and modification May be immunogenic (if viral based) Large BAC/PAC difficult to chemically transduce cells Amplicon Virion packaging Infectious particles Transgene capacity limited by virion packaging capacity Minimal immune response potential Ease of purification and modification Wild-type virus stock contamination

Abbreviations: AC, artificial chromosome; BAC, bacterial artificial chromosome; HAC, human artificial chromosome; PAC, P1-derived artificial chromosome; YAC, yeast artificial chromosome. For an excellent review detailing the advantages and disadvantages of various extrachromosomal vector technologies, see Lufino et al.2

Gene Therapy Human artiﬁcial chromosomes S Macnab and A Whitehouse 1183 ab and inverted human telomeres into circular BACs. On cleavage of the tos site, a linear molecule will be pac ori produced with telomeres in the correct orientation. This N15 methodology would generate large linear DNA produ- protelomerase cing a transgene capacity similar to other HACs, without Transfection of having to perform puriﬁcation and restriction digest HAC-BAC protocols, thereby increasing the production yield and reducing DNA fragmentation.

A YAC-based method for genomic vector construction using TAR cloning Inclusion of required chromosomal Expression of genomic size loci allows for accurate VLP production elements transgene expression as the DNA sequence contains intronic and long-range control elements and features, which are essential for natural levels of protein expression. This is important in the clinical improvement of patients undergoing gene therapy protocols, because of 2 Stable mitotic Stable mitotic the potential silencing of cDNA transgenes. The segregation of segregation of construction of vectors containing and expressing geno- HAC-BAC linear AC mic transcripts can, however, be problematic. This issue has recently been addressed using YAC technology.8 This technique uses TAR cloning and exonuclease chromato- Figure 1 Hybrid technologies. (a) HAC–BAC ampVLPs. a-satellite graphy purification. The protocol relies on the incorpora- DNA (red circles) was cloned into HSV-1 amplicon plasmids tion of genomic fragments, in addition to a TAR-cloning containing an origin of replication (ori) and HSV packaging vector containing a yeast selectable marker and yeast sequences (pac), as well as the transgene of interest (green box). The resulting HAC–BAC are transfected into a helper packaging cell centromeric locus (into yeast spheroplasts) (Figure 2a). line containing an oversized HSVDICP27 helper genome able to Recombination events between the genomic region and produce empty ampVLPs. The packaging signal contained with the TAR vector targeting sequences produce genomic trans- HAC–BAC allows packaging of the plasmid and maturation of the gene containing YACs. Yield of gene-positive clones ampVLPs. The resulting stocks can be delivered to target cells and varies from 1 to 5%. Currently, these vectors have maintained because of presence of a-DNA. (b) Generation of linear applications in functional/comparative genomics, and BACs. The inclusion of a tos site (yellow box) within a BAC plasmid allows for the generation of linear constructs in E. coli post cleavage long-range haplotyping because of the fact that multiple and joining of tos by the N15 protelomerase. Inclusion of a-satellite gene isolates are produced, potentially allowing for both sequences (red circles) and human telomeres (black boxes) should parental alleles to be analysed. Importantly, this system produce a method for producing high quantities of mitotically may be able to efficiently produce vectors containing stable HACs. A full colour version of this figure is available at the functional genes from individuals without a diseased Gene Therapy journal online. genotype, which could then be further propagated for use in gene therapy protocols. months. However, the authors do note that HAC–BACs were unstable in 293 and MRC-5V2 cells. This observed A novel method for BAC transgene instability may be due to natural over-expression of the molecular tagging cancer inducing, anti-apoptotic chromosome regulating proteins AIM-1 and Topo II.15 This highlights the fact that The construction of BAC vectors containing transgenes different HAC constructs may have varied suitability for of interest, as well as the molecular tools for subsequent different cell types and that vector design will have to protein study, such as N- or C-terminal tagging, can compensate for different genetic backgrounds. However, involve many complex cloning strategies, which are time this work presents a significant advance in the field of and resource intensive. Owing to the complexity of HAC delivery. manipulating genomic loci, cDNAs are the method of An additional technology that further blurs the line choice for studying protein interactions. However, cDNA between HAC and BAC vectors is a novel method for transgenes may not provide natural levels of expression linearising circular DNA, though stably maintaining it in or be completely translationally modified, and, therefore, E. coli. The protocol involves inserting a telomeric N15 study of genomic transcripts is preferable. Cloning of telomere occupancy site (tos) fragment into the circular tagged genomic transcripts until recently was technically BAC. The tos site consists of a region of three inverted challenging. However, as genome sequencing projects repeats, which get cleaved and rejoined to form a pair of advance, mapped BAC libraries are being produced for capped telomeric ends on the linear DNA fragment by most model organisms.17,18 Poser et al.18 have developed the N15 protelomerase16 (Figure 1b). The capping of the a pipeline methodology, which generates hundreds of linear DNA fragments inhibits the degradation of the transgenic constructs, and tens of transformed cell lines, DNA by enzymes such as RecA. This technology may per person, per month, termed as ‘BAC TransgenOmics’ 18 provide a tool for studying the requirements of centro- (Figure 2b). This elegant technique uses BAC recombina- mere formation. In addition, generation of linear HACs tion, producing N- or C-terminally tagged genomic could be achieved by the inclusion of a-satellite DNA transcripts contained within a BAC library. Initial

Gene Therapy Human artificial chromosomes S Macnab and A Whitehouse 1184 a Genomic region of b BACs is size independent, as long-term delivery and interest expression of genomic transcripts (4100 kb) in the liver Genomic BAC library has been shown after hydrodynamic mouse tail vein injection.11 Although the authors focused on gene expression in the liver, this technique has been shown TAR vector to deliver BAC-expressing luciferase transgenes to organs such as the heart, lung, kidney, spleen and Molecular tag muscles albeit with a decrease in efficacy. Delivery of BACs is less efficient using this methodology compared with plasmid DNA; however, BAC DNA was found to have enhanced transgene expression compared with plasmid DNA. This research indicates that the large- TransgeneOmics pipeline sized plasmids do not necessarily hinder transfection Yeast efficacies in vivo and that somatic delivery of BACs is a spheroplast viable option and may, therefore, alleviate problems associated with virally mediated gene transfer. This data is surprising and important, as the majority of research reports low efficacies of large construct transfection in vitro.2 Transgene studies

Advances using ACs to deliver a wide Figure 2 Construction of genomic vectors for gene expression. variety of transgenes including CFTR, (a) Gene isolation by TAR in yeast. Genomic DNA fragments are FRDA and dystrophin mixed with an excess of TAR-cloning vector. The vector contains two homology regions (yellow and green pentagons) specific to the BACs are being developed to express a number of gene of interest (blue box). The vector also contains the HIS3 yeast therapeutic genes. Recently, a BAC with a 250 kb insert selection marker and a yeast centromeric locus (CEN6, red circle). was produced by red homologous (g-mediated) recom- The DNA is introduced into yeast spheroplasts. Recombination can 0 0 occur between the TAR vector and the genomic region of interest bination expressing the CFTR gene, as well as 5 and 3 forming a circular DNA molecule, containing the genomic region of regulatory elements. Red recombination uses phage- interest, which is capable of stable propagation in yeast. (b) ‘BAC expressed proteins to cause recombination and is TransgeneOmics’. A molecular marker can be tagged either at the restriction site and sequence size independent, thereby N- or C-terminus of a BAC genomic library. The tag and the BAC making this technique ideal for cloning genomic tran- are combined through the TransgeneOmics pipeline, which consists of a 96-well format temperature sensitive recombination system. scripts. Cystic fibrosis (CF) is a common, fatal genetic Tagged vectors can be produced within a period of 24 h. These disease that affects 1 in 2500 Caucasians and is caused by vectors can then be used to understand the nature of the genomic mutation in the CFTR gene. Transfection of the CFTR- transcript contained with the BAC. For an excellent review of BAC BACs into murine CMT-93 cells showed that full length, TransgeneOmics, see Roguev and Krogan, 2008.17 A full colour correctly spliced CFTR mRNA could be detected after version of this figure is available at the Gene Therapy journal online. chemical transfection. However, these studies are limited to determining mRNA levels, because of the presence of endogenous mouse CFTR, thereby making studies at the cultures are produced in a period of 24 h in a 96-well protein level difficult. However, these vectors still have a format. BAC TransgenOmics is suitable for studying large potential for correcting the phenotype of epithelia protein localisation, tandem affinity purified protein– cells in a CF patient as the genomic transgene undergoes protein interactions and chromatin immunoprecipitation natural splicing events. Although expression of genomic (ChIP) assays. The authors have also shown that this CFTR has previously been achieved in YAC systems, this technology can be used for studying the effects of new CFTR-BAC expression system will allow for ease of proteins during growth and development by applying vector propagation and purification, as well as controlled their BAC TrangenOmic protocol to embryonic stem (ES) and tissue-specific transgene expression.19 However, as cells. These technologies will allow for the easier study of with many protocols concerning CF, delivery of the genomic transcripts and associated proteins, and should, vector is proving to be problematic. Nebulisers are therefore, allow determination of the success of genomic currently used to alleviate symptoms of CF by delivering loci expression of BAC delivered therapies to become a saline, drugs or water to the patient. This technology standardised high throughput technique. may be suitable for use with CFTR-BACs; however, BAC plasmids are very fragile and nebulisers will undoubt- edly require modification to ensure correct cushioning of the BAC during delivery. One method may be to package BAC construct size may not affect the BAC in a viral coat and then use a nebuliser ensuring in vivo vector delivery a physical coat, which will protect the BAC from mechanical stress. Current research has shown that BAC plasmids can HSV-1 amplicons are at the forefront of amplicon successfully express complete genomic loci; however, technology. They are able to deliver and express genomic delivery of these constructs can be problematic. Recent loci in excess of 100 kb, and in vitro analysis shows that studies have shown that somatic in vivo transduction of they are able to rescue phenotypes in cellular models of

Gene Therapy Human artiﬁcial chromosomes S Macnab and A Whitehouse 1185 20 disease. Recent advances in amplicon-derived thera- abcd peutics include delivery and long-term expression of the

135 kb genomic DNA insert containing the FRDA gene, BAC/PAC/YAC Amplicon Viral BAC HAC encoding frataxin. This protein participates in the biogenesis of iron–sulphur clusters. Mutant FRDA genes 1 1 result in Freidreich’s ataxia, which affects 1–2 in 50 000 1 Caucasians. This disease mainly affects the central 2 2 1 nervous system and cardiac tissue, and is characterised by gait, motor-neuron degeneration and cardiac hyper- 2 trophy. Current treatments have had some success in 3 2 3 treating cardiac tissues, but have failed to treat the 21 neurological disorders. The HSV amplicon system 3 4 4 allowed for production of high titre FRDA vectors 3 (B3 Â 107 TU ml–1). Infection of cells with ampVLPs resulted in a significant restoration of the wild-type oxidative stress resistance phenotype in primary 4 5 5 Freidreich’s ataxia fibroblasts in vitro, which are other- 4 wise extremely sensitive to oxidative conditions, suggesting a potential new avenue for gene therapy applications against this disease. Importantly this vector expresses the transgene at physiological condi- 5 tions and has the advantage of using ampVLPs, which minimise host exposure to any adverse viral genes or Vector size proteins. Up to 2.3 Mb Virion capacity Virion capacity Unlimited HACs are also showing promise in the field of molecular therapeutics. A recent study using a mini- Transduction method chromosome-based HAC was shown to immortalise Biochemical VLP delivery Virus infection MMCT targeted cells. This may be beneficial for auto-, allo- Transduction Efficacy 22 and xenotransplantion therapy. The human telomerase 5-10% ~100% ~100% 0.005 % reverse transcriptase transgene is responsible for indu- Target cells (in vitro) cing telomerase activity and stabilising telomere length, Human VLP specific Virus specific Somatic cell thereby increasing the life span of expressing cells. HAC- Animal Embryonic stem cells mediated transduction of human telomerase reverse Human primary fibroblasts transcriptase into human fibroblast cells (HFL-1) ex- Figure 3 Delivery method of ACs. (a) BAC/PAC/YAC plasmids tended their ability to grow in culture for over 50 days, can be delivered through transfection reagents such as PEI. 1–2: The with a 10-fold increase in cell numbers.22 This is the first AC is purified and mixed with a suitable lipid-based biochemical to demonstration of non-integrating ACs increasing the form DNA complexes. 3: Target cells are incubated with DNA proliferative capacity of primary cells. Although success- complex until DNA is transferred to cells. 4: AC has the properties ful, the HAC was transferred to the HFL-1 cells through to be maintained within cell populations when continually expressing transgene. (b) Amplicon delivery. 1: Amplicon plasmids microcell-mediated chromosome transfer (MMCT) are introduced into a helper cell line, which can be cosmid, (Figure 3), and technologies will need to be improved attenuated virus, or wild-type virus based. 2–3: Helper cell line in the field of HAC purification and delivery before produces a helper-free ampVLP stocks. 4: ampVLPs are used to this HAC becomes a viable tool in clinical gene infect target cells and express transgene. 5: Amplicon plasmid can therapy. be maintained within progeny cells. (c) Viral BACs. 1–3: A viral BAC Another recent advance in HAC-mediated therapy is is transfected into a permissive cell line to produce virus particles. 4: Virus stocks are used to infect target cells and express transgene. 5: the development of an HAC vector capable of expressing Depending on virus and cell type, there is potential for virus the entire human dystrophin gene, designed for treat- production or immune stimulation. (d) HAC delivery. 1: HAC are ment of Duchenne muscular dystrophy (DMD).23 DMD produced. 2: Cells containing HACs (small chromosome with green is a recessive X-linked, fatal disorder caused by the centromere) are exposed to drugs such as colcemid to block mitosis. absence of dystrophin protein in skeletal muscle, brain, 3: Nuclear envelopes form around individual chromosomes form- retina and smooth muscle. The disease affects 1 in 3500 ing micro-nucleated cells. 4: Centrifugation in the presence of cytochalasin B, followed by filtration is used to collect micro- males, and eventually leads to paralysis, fatigue and nucleated cells. 5: Recipient cells are fused with the micro-nucleated skeletal deformities. The Dystrophin gene spans 2.4 Mb cell to deliver the HAC. Green box represent transgene of interest and encodes a number of tissue-specific isoforms. Over- (a–c). Small chromosome represents constructed HAC (d). VLP, or under-expression of these isoforms can lead to virus-like particle. A full colour version of this figure is available at different muscular dystrophies. To address the issue of the Gene Therapy journal online. natural, isoform-specific gene regulation, the entire dystrophin gene was cloned, through loxP recombination, into a HAC produced from a truncated human chromosome 21. The resulting DYS-HAC was shown to application as HAC transfection was again achieved be stable in human immortalised mesenchymal stem through the MMCT delivery method and research is cells. In addition, production of chimeric mice containing required to determine a therapeutic proof of principle. the DYS-HAC vector were shown to express the A second group has also managed to show the appropriate isoforms in the correct tissues. However, potential of ACs in the use of DMD gene therapy.24 the DYS-HAC vector still has limitations in its clinical Hoen et al.24 have generated human DMD (hDMD) mice

Gene Therapy Human artificial chromosomes S Macnab and A Whitehouse 1186 with intact and functional copies of the Dystrophin truncated human chromosome 21) and delivered to ES genomic transcript expressed from a YAC through fusion and mGS cells through MMCT. Moreover, chimeric of DYS-YAC-containing yeast spheroblasts with mouse mice were produced using both ES and mGS cells, ES cells. Analysis revealed levels of Dystrophin similar to which on analysis of p53 isoforms showed correct endogenous mouse Dystrophin. Moreover, the transgene splicing in different tissue types. In addition, in vitro was found to be integrated into mouse chromosome 5 studies indicated that HAC-mediated p53 expression and breeding studies provided functional proof of resulted in a 10-fold increase in apoptosis in principle as the hDMD mice were crossed with dystro- X-ray irradiated mGS cells, as well as the ability to phin-deficient mice, which resulted in rescue of the lethal differentiate into different embryonic germ layers after dystrophic phenotype, with all signs of muscular subcutaneous injection into nude mice, in contrast to dystrophy disappearing. In addition, these studies have control mGS cells. These experiments are important produced an animal model, which should allow direct because they again show the potential for AC and stem- research into molecular therapeutics or genetic studies of cell therapeutic both in vivo, ex vivo and in vitro. hDMD. However, as the YAC integrates into the host Furthermore, they may negate ethical concerns over chromosome, this could lead to alteration of natural gene current research practices. expression and genomic stability. These studies provide proof of principle that ACs can successfully be used to deliver and express large ACs have limitations in delivery and genomic constructs. In addition, ACs expressing geno- production mic transcripts can produce the natural splice variants for each targeted tissue, something cDNA templates Each vector system has its limitations regarding their cannot achieve. This is important not only for dystrophin usefulness as an AC. BAC vectors are limited by their and CFTR, which have complicated regulation profiles total transgene capacity, which although is not insignif- requiring appropriate levels of functional isoforms to be icant, cannot match the potential of YACs and HACs produced, but also for other genomic transcripts, which (Table 1; Figure 3). Transgene capacity and delivery have equally complex expression profiles. mechanisms of BACs are being improved through the development of ampVLPs; however, these technologies are still hampered by the immunogenicity, which can be ACs and stem-cell-based therapies generated from associated viral gene expression, cellular debris or wild-type virus contamination. These issues Investigations are currently ongoing to determine the will need to be addressed before these vectors are potential of ACs for combined use with stem-cell deemed to be truly safe. therapeutics. One area of particular interest is the use HAC technology seems to be the most natural of AC-containing stem cells as gene delivery vechicles.25 mechanism for expressing and maintaining transgenes; Kinoshita et al.25 have recently used human immortalised however, this technology is severely hampered in its mesenchymal stem cells containing an HAC expressing clinical application through low transfection efficacies the herpes simplex virus thymidine kinase (tk) gene to and purification technologies. The exception is satellite show that gene delivery causes a significant reduction in DNA-based HACs (SATACs). These HACs are generated glioma mass compared with controls after ganciclovir by inclusion of a selectable marker and recombinant treatment in nude mice models. DNA into nuclear organisation regions and can be ACs can also be used in ex vivo stem-cell therapeutics.26 transfected using lipid-based reagents at efficacies, Katona et al.26 have recently shown ex vivo delivery of typically 0.1–1% or by the use of cationic lipids and an AC expressing mouse or human derivatives of the high-frequency ultrasound (typically 12–53%, depending galactocerebrosidase transgene through transfection on cell type). To date, other forms of HACs can only be into mouse ES cells. Galactocerebrosidase deficiency is delivered to the target cell by the use of whole cell fusion responsible for Globoid cell leukodystrophy (also or MMCT.1,2,14 Purification is also problematic for HACs. known as Krabbes’s disease), which is associated with Work is ongoing to develop protocols for HAC purifica- demyelination, severe gliosis and the presence of globoid tion using flow cytometery. Currently, only murine- cells. It is currently hypothesised that cell replacement based SATACs have been successfully purified by flow therapy will be the best course of treatment for this cytometery. Features of these murine ACs include a large genetic disease. To that end, chimeric mice were size (over 40 Mb in length) and a high satellite DNA produced from ES cells, which expressed the galactocer- content, which allows ease of identification and purifica- ebrosidase transgenes. Experimentation revealed that the tion through staining techniques. However, the composi- life span of chimeric mice was increased twofold, tion of murine-based SATACs is typically different from showing the potential of ex vivo AC stem-cell therapies other HACs that are under development, which are in the treatment of reversible genetic diseases and much smaller, have a lower satellite DNA composition cancers through the production of modified replacement and are consequently difficult to distinguish from cells. nuclear material. In addition, only murine-based SATAC Although highly promising, ES-cell research is fraught chromosomes thus far have been reported to be routinely with ethical issues. One proposed methodology to purified at reasonable rates (95% purity, 1 million vectors negotiate this issue is the use of multipotent germline per hour).1 Purification of high quantities of HACs will (mGS) cells.27 Recent work by Kazuki et al.27 has prove to be a major limiting factor before they can be shown functional restoration of a genetic deficiency in routinely used in gene therapy applications. murine p53À/À mGS cells. This was achieved by Another potential problem associated with HACs is delivering a human p53 expressing HAC (based on a the large vector size. Most vectors are limited by the size

Gene Therapy Human artificial chromosomes S Macnab and A Whitehouse 1187 of transgene that can be incorporated into the vector of Conflict of interest choice. However, the opposite may be true for HACs. Studies performed in yeast suggest that DNA molecules The authors declare no conflict of interest. that are one-fifth the size of the smallest natural chromosome are a minimum for stability during mitosis. Therefore, HACs may have to be B10 Mb to be References mitotically stable. This hypothesis is reinforced by experiments carried out showing that HACs of B2– 1 Duncan A, Hadlaczky G. Chromosomal engineering. Curr Opin 4 Mb vary in their ability to be mitoically stable, whereas Biotechnol 2007; 18: 420–424. HACs closer to 10 Mb maintained their ability to under- 2 Lufino MM, Edser PA, Wade-Martins R. Advances in high- go mitotic segregation to a high fidelity. This presents a capacity extrachromosomal vector technology: episomal main- problem for the generation of HAC vectors, as a tenance, vector delivery, and transgene expression. Mol Ther substantial amount of non-coding or ‘stuffer’ DNA 2008; 16: 1525–1538. may be required if only a single genomic transcript is 3 Ren X, Tahimic CG, Katoh M, Kurimasa A, Inoue T, Oshimura being used in a therapeutic protocol. However, the choice M. Human artificial chromosome vectors meet stem cells: new prospects for gene delivery. Stem Cell Rev 2006; 2: 43–50. of stuffer DNA must be carefully considered, as 4 Monaco ZL, Moralli D. Progress in artificial chromosome transgene expression studies using Adenovirus ampli- technology. Biochem Soc Trans 2006; 34: 324–327. con systems have revealed that transgene expression 5 Heaney JD, Bronson SK. Artificial chromosome-based trans- levels are affected by the type of stuffer DNA incorpo- genes in the study of genome function. Mamm Genome 2006; 17: rated into the vector. 791–807. 6 Peterson KR. Preparation of intact yeast artificial chromosome DNA for transgenesis of mice. Nat Protoc 2007; 2: 3009–3015. Prospects 7 Leem SH, Yoon YH, Kim SI, Larionov V. Purification of circular YACs from yeast cells for DNA sequencing. Genome 2008; 51: As YACs stimulated ideas into the field of ACs, it is, 155–158. therefore, rather fitting that there has been a flurry 8 Kouprina N, Larionov V. Selective isolation of genomic loci from of developments to purify YACs at reasonably high complex genomes by transformation-associated recombination yields. YACs, therefore, present an interesting prospect cloning in the yeast Saccharomyces cerevisiae. Nat Protoc 2008; 3: as gene therapy vectors in the clinic, as well as being 371–377. commercially viable. However, like HACs, improvement 9 Zhou F, Li Q, Gao SJ. A sequence-independent in vitro in the methods for delivery to target cells are still transposon-based strategy for efficient cloning of genomes of required. large DNA viruses as bacterial artificial chromosomes. Nucleic HACs have the ability to express multiple exogenous Acids Res 2008; 37: e2 (doi:10.1093/nar/gkn890). transgenes, in a non-integrated state, that uses natural 10 Macnab S, White R, Hiscox J, Whitehouse A. Production of an cellular mechanisms of chromosomal maintenance. The infectious Herpesvirus saimiri-based episomally maintained amplicon system. J Biotechnol 2008; 134: 287–296. recent success of HAC-mediated expression of large 11 Hibbitt OC, Harbottle RP, Waddington SN, Bursill CA, Coutelle genomic transgenes suggests that these constructs will be C, Channon KM et al. Delivery and long-term expression of a suitable as gene therapy vectors in vivo. Furthermore, a 135 kb LDLR genomic DNA locus in vivo by hydrodynamic tail significant step in delivering HACs to target cells has vein injection. J Gene Med 2007; 9: 488–497. been taken by the development of the amplicon-based 12 Griffiths RA, Boyne JR, Whitehouse A. Herpesvirus saimiri- 14 HAC. This technology is important because it combines based gene delivery vectors. Curr Gene Ther 2006; 6: 1–15. the infectious traits of the ampVLP, with the biosafety of 13 Grant KG, Krisky DM, Ataai MM, Glorioso III JC. Engineering an HAC, allowing for improvement in the delivery of cell lines for production of replication defective HSV-1 gene HAC vectors. therapy vectors. Biotechnol Bioeng 2008; 102: 1087–1097. Amplicon technology incorporating viral episomal 14 Moralli D, Simpson KM, Wade-Martins R, Monaco ZL. A novel maintenance elements and VLPs, presents a promising human artificial chromosome gene expression system using avenue of research for delivering and maintaining ACs. herpes simplex virus type 1 vectors. EMBO Rep 2006; 7: Moreover, these systems are showing great potential for 911–918. expressing small genomic transcripts, providing cheap 15 Jiao W, Lin HM, Timmons J, Nagaich AK, Ng SW, Misteli T et al. and quick methods of production and purification. E2F-dependent repression of topoisomerase II regulates hetero- Moreover, they are able to effectively target a wide chromatin formation and apoptosis in cells with melanoma- variety of cells. However, further research is still prone mutation. Cancer Res 2005; 65: 4067–4077. required to remove wild-type contamination from 16 Ooi YS, Warburton PE, Ravin NV, Narayanan K. Recombineer- ampVLP stocks. ing linear DNA that replicate stably in E. coli. Plasmid 2008; 59: The recent developments in AC technology present 63–71. 17 Roguev A, Krogan NJ. BAC to the future: functional genomics in improved methods for the production, purification, mammals. Nat Methods 2008; 5: 383–384. delivery and natural transgene expression of genomic- 18 Poser I, Sarov M, Hutchins JR, Heriche JK, Toyoda Y, sized loci. These technologies are all steps forward in Pozniakovsky A et al. BAC TransgeneOmics: a high-throughput alleviating problems associated with synthetically pro- method for exploration of protein function in mammals. Nat duced cDNA. In addition, AC technology is proving to Methods 2008; 5: 409–415. be highly compatible with stem-cell research. With 19 Kotzamanis G, Abdulrazzak H, Gifford-Garner J, Haussecker further development, ACs could be used to improve PL, Cheung W, Grillot-Courvalin C et al. CFTR expression from a the efficacy of gene therapy by providing physiologically BAC carrying the complete human gene and associated appropriate expression of transgenes in vivo in target regulatory elements. J Cell Mol Med 2008; 24 (Postprint; tissues. 10.1111/j.1582-4934.2008.00433.x).

Gene Therapy Human artificial chromosomes S Macnab and A Whitehouse 1188 20 Hibbitt OC, Wade-Martins R. Delivery of large genomic DNA 24 Hoen P, de Meijer E, Boer J, Vossen R, Turk R, Maatman R et al. inserts 4100 kb using HSV-1 amplicons. Curr Gene Ther 2006; 6: Generation and characterization of transgenic mice with the full- 325–336. length human DMD gene. J Biol Chem 2008; 283: 5899–5907. 21 Gomez-Sebastian S, Gimenez-Cassina A, Diaz-Nido J, Lim F, 25 Kinoshita Y, Kamitani H, Mamun M, Wasita B, Kazuki Y, Wade-Martins R. Infectious delivery and expression of a 135 kb Hiratsuka M et al. A gene delivery system with a human human FRDA genomic DNA locus complements Friedreich0s artificail chromosome vector based on migration of mesenchy- ataxia deficiency in human cells. Mol Ther 2007; 15: 248–254. mal stem cells towards human glioblastoma HTB14 cells. Neurol 22 Shitara S, Kakeda M, Nagata K, Hiratsuka M, Sano A, Osawa K Res 2009 (doi:10.1179/174313209X455718). et al. Telomerase-mediated life-span extension of human 26 Katona R, Sinko I, Hollo G, Szucs K, Praznoszky T, Kereso J et al. primary fibroblasts by human artificial chromosome (HAC) A combined artificialchromosome-stem cell therapy method in a vector. Biochem Biophys Res Commun 2008; 369: 807–811. model experiment aimed at the treatment of Krabbe’s disease in 23 Hoshiya H, Kazuki Y, Abe S, Takiguchi M, Kajitani N, Watanabe the Twitcher mouse. Cell Mol Life Sci 2008; 65: 3830–3838. Y et al. A highly stable and nonintegrated human artificial 27 Kazuki Y, Hoshiya H, Kai Y, Abe S, Takiguchi M, Osaki M et al. chromosome (HAC) containing the 2.4 MB entire human Correction of a genetic defect in multipotent germline stem cells dystriophin gene. Mol Ther 2008; 17: 309–317. using a human artifical chromosome. Gene Therapy 2008; 15: 617–624.

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