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New method of gene therapy used to treat hemophilia B in mice
By Bijal P. Trivedi

Researchers have used a new type of gene therapy to treat mice with the rodent equivalent of hemophilia B. The gene entered five to six percent of liver cells and produced clotting protein for more than nine months, essentially curing the disease.

Millions of copies of the human Factor IX clotting gene (red) and the transposase gene (green) are injected into the tail of the mouse.

The team used a human clotting gene called human factor IX (hFIX), which is lacking in hemophelia B, and added it to a larger piece of DNA called a transposon that can integrate into the chromosome.

"The advantage of the gene-transposon combination is it integrates into the chromosome and continues to produce the protein for the life of the cell," says Mark Kay of Stanford University School of Medicine and an author of the study which appears in the May issue of Nature Genetics. When the cell divides, the transposon is replicated along with the rest of the DNA and passed to both cells.

In the nucleus of a liver cell the transposase gene makes a transposase protein (yellow) which releases the hFIX gene.

Transposons are pieces of DNA that can move from one location to another with the help of an enzyme called transposase. Kay's groups administer the genes by injecting millions of copies of the two pieces of DNA, one with the transposase gene and the other with the transposon containing hFIX, into the tail of the mouse. When the two pieces of DNA enter the same cell, the hFIX gene inserts itself into the chromosome. The mice were tested 102 days later to see whether the hFIX gene was producing the blood-clotting factor.

The hFIX gene integrates into the chromosome.

Researchers found that hemophiliac mice treated with the hFIX and transposase genes formed clots in less that seven minutes, indicating that the human clotting factor was being produced. Normal healthy mice are able to form blood clots within three minutes. Untreated mice with hemophilia B could not form clots at all.

This method of gene therapy differs from other approaches because it injects 'naked' DNA directly into the body, rather than placing it in some type of capsule. It may prove a better method in the long run. After the first dose of virus-mediated gene therapy the body's defenses recognize the virus, and second or third doses are subsequently destroyed by the immune system, destroying the therapeutic genes in the process, says Kay. Transposon therapy does not appear to trigger any immune reactions but further experiments must be done to prove this, he added.

Critical future experiments involve testing to determine that once the transposon has integrated into the chromosome, it won't "jump out," says Kay. "We need to make sure that it is a dead-end event." Experiments must also be done to show that the transposase doesn't cause other genes to jump out or rearrange the chromosomes.

In addition, Kay will test whether repeated doses of the transposon therapy can be used to boost the number of cells that carrying the gene.

The transposon system is also a useful biological tool for introducing new genes into mice and studying the effect of the new gene throughout their lives.

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Yant, S.R. et al. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 25, 35-41 (May, 2000).

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