A novel technique for creating human artificial chromosomes

Human artificial chromosomes (HACs) capable of working within human cells could power advanced gene therapies, including those addressing some cancers, along with many laboratory applications — but serious technical obstacles have hindered their development. Now a research team led by the Perelman School of Medicine at the University of Pennsylvania has explained how they devised an efficient technique for making HACs from single, long constructs of designer DNA.

The first HACs were developed 25 years ago, and artificial chromosome technology is already well advanced for the smaller, simpler chromosomes of lower organisms such as bacteria and yeast. Human chromosomes are another matter, due largely to their greater sizes and more complex centromeres, the central region where X-shaped chromosomes’ arms are joined.

Researchers have been able to get small artificial human chromosomes to form from self-linking lengths of DNA added to cells, but these lengths of DNA tend to join together, or ‘multimerise’, with unpredictable organisations and copy numbers — complicating their therapeutic or scientific use — and the resulting HACs sometimes even end up incorporating bits of natural chromosomes from their host cells, making edits to them unreliable. The new method, described in the journal Science, allows HACs to be crafted more quickly and precisely, which will speed up the rate at which DNA research can be done.

“Essentially, we did a complete overhaul of the old approach to HAC design and delivery,” said Ben Black, a professor at Penn. “The HAC we built is very attractive for eventual deployment in biotechnology applications, for instance, where large-scale genetic engineering of cells is desired. A bonus is that they exist alongside natural chromosomes without having to alter the natural chromosomes in the cell.”

The HACs devised by the Penn research team included larger initial DNA constructs containing larger and more complex centromeres, which allow HACs to form from single copies of these constructs. For delivery to cells, they used a yeast-cell-based delivery system capable of carrying larger cargoes.

“Instead of trying to inhibit multimerisation, for example, we just bypassed the problem by increasing the size of the input DNA construct so that it naturally tended to remain in predictable single-copy form,” Black said. This method was much more efficient at forming viable HACs compared to standard methods, and yielded HACs that could reproduce themselves during cell division.

The potential advantages of artificial chromosomes — assuming they can be delivered easily to cells and operate like natural chromosomes — are many. They would offer safer, more productive and more durable platforms for expressing therapeutic genes, in contrast to virus-based gene-delivery systems which can trigger immune reactions and involve harmful viral insertion into natural chromosomes. Unlike relatively cramped viral vectors, artificial chromosomes would permit the expression of large, cooperative ensembles of genes, for example to construct complex protein machines. Furthermore, Black expects that his group’s approach will be useful in making artificial chromosomes for other higher organisms, including plants for agricultural applications such as pest-resistant, high-yield crops.

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