Artificial Chromosomes: Care for a Pair?
⌘ How about a new pair of chromosomes to add to the 23 pairs you already own? They make a great vehicle to insert new genes to reprogram cells, to replace defective or damaged genes. Current forms of gene therapy use modified viruses to deliver a stripped down version of a gene into cells. But these minigenes are hard to control. They may insert randomly into the chromosome and disrupt some essential function (“insertional mutagenesis”), trigger undesired immune response to the viral carrier, or rarely, activate a cancer-causing gene. Artificial chromosomes offer an alternative system of gene delivery.
⌘ Of YACs, BACs and Life HACs: Yeast artificial chromosomes (YACs) were first introduced 30 years ago, followed by bacterial versions (BACs) which are used in research as a convenient way to clone and sequence DNA from other genomes. More recently, human artificial chromosomes (HACs) have been successfully introduced into mice to cure Duchenne muscular dystrophy (http://goo.gl/xuvSS).
⌘ How to Build a HAC: You probably know that DNA combines with proteins (called histones), like pearls on a string, which are then packaged and condensed into a chromosome. Chromosomes have short repeating fragments of DNA sequence, known as alpha satellites that can stretch for millions of base pairs, to form a centromere (the central knot of the X-shaped chromosome in the image below). The tips of the chromosome are capped with other protective repeating units called telomeres. Then there are the origins of replication, which are designated start sites for copying. An artificial chromosome has all these features too. It can be made by a top down approach: small fragments of telomere sequence (TTAGGG)n are introduced into a cell where they insert into chromosomes, triggering the loss of all sequences distal to the point of insertion. This eventually whittles down a chromosome until only a functional stump, about a tenth the size of a normal chromosome, remains. Alternatively, they can be assembled by a bottom up approach starting with de novo assembly of blocks of alpha satellite DNA. Entire genes, such as the large dystrophin gene defective in muscular dystrophy, can now be inserted, using special targeting sites (loxP).
⌘ Why are they better? Because the artificial chromosome has a centromere tethering it to the mitotic spindle during cell division, it can be partitioned into newly divided cells to survive stably in the long term. Second, there is no upper size limit to DNA cloned in a HAC: a gene with all its neighboring regulatory elements can be used so as to faithfully mimic the natural pattern of gene expression. In fact, groups of genes encoding complex pathways can be carried on a single HAC. Third, because of the lack of viral sequences, HAC vectors minimize harmful immune responses in the host and the risk of triggering cancers.
⌘ Scientist Gregory Stock thinks it may be another 20 years before we see artificial chromosomes put to use in humans. For now, artificial chromosomes are still difficult to introduce into cells, with efficiencies as low as 1 in 10,000. “Bioengineers tend to underestimate the complexity of human biology,” he says. “These developments often come at a slower pace than we imagine. But they’re inexorable.”
▶ Image and Pop Sci story on the future of HACs: http://goo.gl/FfE5c
▶ Reference Paper on HAC (open access and easy to read introduction): http://goo.gl/cmkla