The use of human artificial chromosomes (HACs) has advanced over the past 25 years. Yet, despite advances in technology to produce and use HACs, there are still many hurdles to overcome before they are useful for therapeutic applications.

An international team of researchers from the University of Pennsylvania and the University of Edinburgh constructed single-copy HACs to address the issue of multimerization. Craig W. Gambogi, PhD, University of Pennsylvania, and colleagues published their paper, “Efficient formation of single-copy human artificial chromosomes” in the latest issue of Science.

Budding yeast contain native centromeres that function through a 125-base pair DNA sequence, however, the human centromere is much larger and epigenetic. Utilizing centromere epigenetics has helped HAC formation, but does not itself prevent multimerization of the HACs in cells.

HACs have the potential to insert large amounts of engineered DNA into cells, however their construction can be hindered by multimerization. This phenomenon coupled with the rearrangement of the accompanying input DNA during critical early stages of HAC formation “has severely hindered their development toward the broader promise of synthetic biology and therapeutic applications,” the authors wrote.

In a Perspective article published in the same issue of Science, R. Kelly Dawe, PhD, University of Georgia, commented: “Future applications of HACs will likely focus on introducing long genes or multigene clusters into cell lines or individuals. It may soon be possible to include artificial chromosomes as a part of an expanding toolkit to address global challenges related to healthcare, livestock, and the production of food and fiber.”

The new report describes a method that efficiently forms single-copy HACs by employing a large, roughly ~750-kilobase (kb) construct containing multiple chromatin types found in the inner and outer centromere, reducing the need to multimerize. “Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells,” the authors stated.

The researchers circumvented multimerization of centromeres in their HACs by using a technique called protein tethering. They further improved the transfer of HACs from budding yeast to human cell culture by use of cell fusion which showed a reduced instance of multimerization, but also increased efficiency in HAC transfer to cells with the removal of purification steps and allowed for the use of larger HACs. The authors attested: “The central innovation with YAC-Mm-4q21LacO is that it avoids the rampant multimerization that has complicated past HAC efforts and that it permits rapid assessment of HAC function.”

These advances in technique and methodology allowed a relatively large chromosome assemblage in the HAC to be inserted into human cultured cells. The team created HACs with about 760 kb DNA, while other vectors, including those produced in bacteria are limited to about 200 kb.

Though these advances are important steps in the field of HACs, more work is needed to confirm the stability and fecundity of these HACs through cell replication cycles.

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