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| This article will appear in a forthcoming issue of Trends in Molecular Medicine. | |
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Abstract
"Gene therapy" is the use of DNA to correct a genetic defect at the DNA level. Over the years, much has been written about the great promise of this form of treatment, and the ethical issues surrounding it. Despite evidence of measurable success [1,2], gene therapy has in recent times attracted increasing scepticism because of its failure to deliver its promise 15 years of intensive research. A recent fatality attributed to an experimental gene therapy protocol has also raised serious concern [3]. Clearly, the day of gene therapy is not here yet, and more focused research will be required to develop, above all, efficient and safer gene-delivery systems [4].
Conceptually, it is probably not difficult to imagine that a suitably engineered human chromosome, which would carry no unnecessary sequence except for those directly required for therapy and stable propagation in a cell, might constitute an excellent gene-delivery vector system. Why? First, such a vector should be welcomed as chromosome number 47 by our cell. Because "cousin-47" can exist autonomously, it would not be disruptive or mutagenic to the genome, such as can happen with mechanically or virally delivered naked DNA that requires integration into host chromosomes for replication and maintenance. Second, the risk of infectivity or immune reaction associated with the use of conventional viral vectors would be minimal because only naturally occurring human DNA would be used in the chromosome engineering process. Third, chromosomal vectors have a large size capacity, which means that very large genes or gene complexes can be accommodated.
Human Engineered Chromosomes (HECs)
There are two basic strategies to construct a human chromosome vector (figure 1). The so-called "bottom-up" approach aims to assemble a new chromosome de novo from its constituent DNA elements, and the product is commonly referred to as an artificial chromosome. The "top-down" approach starts with an existing human chromosome, which then becomes experimentally reduced in size to a minichromosome. For convenience, the products generated by both these strategies are referred to collectively as human engineered chromosomes (HECs).
The minimum components that a successful HEC needs to have are: (1) sequence motifs that signal DNA replication, necessary for the self-propagation of the chromosome; (2) a centromere, which is essential for the accurate segregation of the replicated sister chromatids to daughter cells; and (3) telomere sequences at both ends of a linear chromosome to provide structural stability to the chromosome ends. Because most genomic DNA pieces larger than 20-30 kb carry some origins of replication, an HEC of the size discussed here (table 1) should automatically contain these motifs. Similarly, telomeres have well-defined sequences, are relatively small in size (in the range of several to tens of kilobases) and do not present a major problem in HEC construction. Furthermore, several presumed circular HECs have been described that can function well without detectable telomere sequences. The centromere is unusually complex, and despite extensive research into its structure, remains enigmatic and a major challenge to the HEC workers.
Bottom-up Construction of HEC
The bottom-up strategy uses, as a centromere, the 171-bp a-satellite DNA that is normally found as long tandemly repeated tracks on human chromosomes. Scientists throughout the US, UK, and Japan have successfully combined synthetic or cloned a-satellite DNA with telomere DNA and, in one study, also exogenous total genomic sequences, to construct artificial chromosomes de novo in a human fibrosarcoma cell line [5-8]. More recently, researchers have incorporated a ~90-kb human HPRT gene into an artificial chromosome and expressed the gene in this cell line [9]. Although these studies have provided important proof-of-concept for the construction of gene-expressing artificial chromosomes, the strategy has several limitations. The most serious is the uncontrolled mode of artificial chromosome formation inside the cell, which makes the structure of these artificial chromosomes highly complex and difficult to define. Often, the transfected DNA will randomly integrate into the genome and undergo amplification and rearrangement, thus severely disrupting the host genome. The usefulness of this method on other cell lines is also unclear.
In a substantial variation to this approach, researchers in Hungary and Canada have collaborated to target large amounts of exogenous DNA into pericentromeric satellite regions and induce large-scale amplification and breakage, to generate a human satellite-associated artificial megachromosome [10]. These artificial chromosomes are generally very big, contain large quantities of a complex combination of different centromeric, pericentromeric and exogenous DNA sequences, and therefore have the same drawbacks as the smaller artificial chromosomes.
Top-down Construction of HECs
In the top-down strategy, a pre-existing chromosome is progressively truncated in situ around its centromere region to remove non-essential chromosome-arm materials. This can be achieved by introducing a new telomere sequence into a truncation site using a DNA construct that contains a telomere sequence, as well as specific genomic DNA to direct homologous recombination at a chosen chromosomal site. Scientists in the UK have reported the generation of several minichromosomes from the human X and Y chromosomes [12,13]. These minichromosomes carry an abundance of a-satellite DNA and are mitotically stable.
Researchers in Australia have been investigating a variant form of human centromere, known as neocentromere [14]. These neocentromeres are derived from non-centromeric genomic sequences and can function normally without any a-satellite DNA. These researchers have recently succeeded in the construction of structurally and mitotically stable neocentromere-based minichromosomes [15].
Choosing HECs
An important criterion for a good HEC for gene therapy studies is that it should have a fully predictable and definable structure. A minichromosome generated by the top-down truncation strategy, by virtue of its better-controlled mode of formation, would satisfy this criterion more so than the de novo assembled artificial chromosomes. Nonetheless, for any of the a-satellite DNA-based minichromosome derivatives, technical difficulty would prevent full-scale sequencing of the large tracks of a-satellite DNA and its immediately flanking genomic regions. In this regard, a fully sequencible neocentromere-based minichromosome will have the advantage of providing a structurally better defined HEC vector.
The size of a HEC is an important consideration, as smaller HECs will clearly be easier to characterize, manipulate and deliver into cells. It is unclear which approach will produce the smallest stable HEC. The choice of a HEC will also depend on how readily therapeutically useful genes can be incorporated (e.g. using the "Cre-loxP"-mediated recombination system), and whether the control of gene expression is in any way compromised. The extent to which the heterochromatic nature of a-satellite DNA or the neocentromerization process16 has on gene silencing also needs to be carefully ascertained and compared.
Gene Therapy Beyond HEC Construction
Given that first-generation HECs can now be built, the usefulness of the HEC technology for gene therapy will, in all probability, rely heavily on whether efficient methods to isolate and deliver these large molecules intact into cells can be developed. Fluorescent-activated cell sorting has been used to purify some of the larger HECs, made possible by their high satellite DNA and chromosome staining properties [17]. Whether the smaller HECs can be similarly isolated remains to be investigated. An alternative method of isolating HECs involves the encapsulation of individual chromosomes in microcells [18]. Several methods have been tested to reintroduce isolated HECs or microcells into target cells. These include lipofection [19], microinjection [20] and/or microcell fusion [18,21,22]. Although these methods have demonstrated varying degrees of success, none of them delivers with good efficiency or precludes the need for extensive screening of cells. Optimization of these methods is needed, especially in terms of their suitability for different cell types. In particular, HEC delivery to primary cell cultures needs to be optimized, based on which initial gene therapy study is being investigated. It is also essential to have good data on the structural and functional integrity of the HECs after transfer into cells, and any side effects the procedures might have on cell phenotype or gene expression.
The problem of low transfer efficiency could potentially be circumvented if a target tissue can be regenerated from a small number of successfully treated cells that can be propagated in culture. An exciting future extension of the HEC technology is to combine it with the treatment of stem cell lines established from reprogrammed somatic cells of a patient to provide an unlimited source of non-immunoreactive tissue materials [23,24].
The development of a good mouse model will allow feasibility studies to be performed, particularly in relation to HEC stability, gene expression, and safety issues in the whole animal. Transgenic mice carrying human chromosomes, or rodent or human artificial chromosomes, have already been produced via microcell transfer into embryonic stem cells [18,22], or by direct pronuclear microinjection [20]. Notwithstanding the limited application of this approach for human gene therapy because of its potential to alter the germline, it has demonstrated that exogenous chromosomes can be delivered and stably maintained in the somatic cells of an animal. In one case, tissue-specific expression of genes on the transferred chromosome has been observed [18].
What is the future of HECs for gene therapy? Present data suggest that we can perform most of the steps outlined in figure 1, albeit inefficiently for some of these steps [25]. Major improvement, in terms of both technical innovation and knowledge-based breakthroughs, is still necessary. Whether we can achieve the level of efficiency of HEC construction and delivery for this technology to be attractive to the gene therapists awaits to be seen. But if we keep in mind the considerable amount of good groundwork that has already been laid, together with the numerous theoretical advantages offered by a HEC vector system, then it might not be so difficult to convince researchers and industries to dig their heels in to face up to the challenges that remain ahead.
Artificial Chromosomes: Ideal Vectors? - a review of progress. From Trends in Biotechnology, 2000, 18:5:218-223. Full text available from BioMedNet.
Gene Therapy: The First Decade - summarizes the approaches taken, results achieved, lessons learned, and important recent developments. From Trends in Biotechnology, 2000, 18:3:119-128. Full text available from BioMedNet.
Centromeres: The Missing Link in the Development of Human Artificial Chromosomes - a review of recent work. From Current Opinion in Genetics & Development, 1998, 8:219-225. Full text available from BioMedNet.
Artificial Chromosomes Coming to Life and Principles for Human Gene Therapy Studies - two recent articles from Science magazine. Registration required for full text.
Delivering the Goods, Gene Therapy's Trials and Tribulations, Gene Therapy Soldiers On, and Genomes and Gene Therapy - four recent articles on gene therapy. From The Scientist.
Introduction to Gene Therapy - includes molecular biology background information, genetic transfer technology, applications, and additional resources. From a course taught at Vanderbilt University.
Institute for Human Gene Therapy - provides news, information, and resources on gene therapy. From the University of Pennsylvania.
Gene Therapy - a journal Web site that allows free access to tables of contents and abstracts, but requires registration for the full text of articles.
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