Contacts: Perry Hackett, Genetics and Cell Biology Department, (612) 624-6736 Deane Morrison, University News Service, (612) 624-2346, [email protected]
U of Minnesota Develops New Way to Get Genes into Chromosomes; Work Addresses Major Hurdle in Gene Therapy
The art of gene therapy could be likened to the art of giving medicine to a child. In both cases, preparing the medicine is often a cinch compared to getting the patient to swallow it. With children, a little sugar may make the medicine go down. But how does one convince a cell to absorb a repaired gene and incorporate it into a chromosome? Currently, two main means of gene delivery are in use. The first, injecting DNA into cells or mixing it with substances that can penetrate cells, generally gets poor results. Little DNA gets incorporated into chromosomes, and those genes that do get incorporated produce spotty output. The second and more widely used method involves attaching genes to viruses, which are good at transporting DNA into cells. Viruses, however, are often destroyed by the patient's immune system, and if they do get into cells, they may cause illness. Further, the viruses used most often can only get into the cell nucleus when the cell is getting ready to divide.
Now, a new method of gene delivery has been developed by University of Minnesota researchers. Working with mobile pieces of DNA known as transposons, the team has found a way to insert any desired DNA sequence into the chromosomes of vertebrate cells with a higher frequency of success than achieved by conventional techniques. The new technique may one day allow doctors to insert working copies of normal genes into the chromosomes of cells to correct disorders caused by faulty forms of the genes. The work will be published in the Nov. 14 issue of Cell.
"We have to have a system to deliver genes to cells," said Perry Hackett, professor of genetics and cell biology at the university. "We believe we've developed a nonviral system that can be used to enhance integration of foreign, or 'trans-,' genes, into human chromosomes. It may be an alternative to using viruses." Hackett's collaborators were Zsuzsanna Izsvk and Zoltn Ivics of the Netherlands Cancer Institute, who did the main work of constructing the new gene delivery system.
In beginning the work, Hackett asked whether vertebrates possessed anything like the transposons that have been found in fruit flies. A transposon is basically just a gene for an enzyme called a transposase. The transposase has only one mission: to cut its own gene free from a chromosome and insert it into another chromosome or at another site on the same chromosome. The transposase recognizes its own gene by the special stretches of DNA--called, appropriately enough, recognition sequences--on either side of the gene. The transposase cuts the gene, recognition sequences and all, and moves the whole package to a new location.
Hackett's group, authorities on fish molecular genetics, studied the genetic makeup of several fish in the salmon family. They found recurring patterns of DNA that suggested that ancestors of the fish had had transposons, but that both the transposase genes and their recognition sequences had mutated to the point where they were no longer functional. It had been, they estimated, at least 15 million years since transposons took their last leaps among the chromosomes of the fish.
After intense study of those ancient DNA sequences, Hackett and his team used statistical methods to figure out which were probably present in the ancient functioning transposon system. They used those pieces to construct a transposase gene and recognition sequences and used the gene to make the transposase enzyme. At this point, they had a gene and an enzyme that could insert the gene in chromosomes. Because they had "resurrected" the transposon system after a sleep of 15 million years, Izsvk named their new system Sleeping Beauty.
To test Sleeping Beauty, the researchers replaced the transposase gene with genes for proteins that could readily be traced--for example, genes for a fluorescent protein or for antibiotic resistance. When they treated cells of zebrafish (a common experimental animal for geneticists) and humans, they found that the cells readily incorporated the foreign genes. These results indicate, said Hackett, that Sleeping Beauty will make a good system for gene transfer in most vertebrates. With further development, Sleeping Beauty could be used to transport normal genes into cells containing defective forms of the genes that cause conditions such as hemophilia or cancer.
"Sleeping Beauty has the potential to revolutionize nonviral approaches to gene transfer and gene therapy," said Scott McIvor, director of the university's Gene Therapy Program. "Sleeping Beauty promises to provide an increased frequency of stable gene transfer. There are many gene therapy companies exploring nonviral approaches."
"The university has assembled a group of scientists who are providing more data on the technology," said Grace Malilay, a patent attorney in the university's Office of Patents and Technology Marketing. "By the end of the month, we expect to form a company to develop the technology. Professor Ari Mukherji of the Carlson School of Management and a team of business students led by Sean Cusick are working with me to put together a business plan." Sleeping Beauty also could be used to investigate the functions of genes.
"Sleeping Beauty can mutate genes by integrating into a chromosome and disrupting the genes it lands in," said Hackett. "We can use the transposon system to isolate and identify genes that cause developmental defects in animals. Many of these same genes will have related genes in humans that are responsible for diseases and developmental problems in children."
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