breakthrough in gene therapy for muscle cells!

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Breakthrough in gene therapy News-Medical.net Thursday, 3-Jun-2004

Despite a roller-coaster ride of ups and downs during the past 15 years,

gene therapy has continued to attract many of the world's brightest

scientists. They are tantalized by the enormous potential that replacing

missing genes or disabling defective ones offers for curing diseases of

many kinds.

One group, consisting of researchers from the University of Wisconsin

Medical School, the Waisman Center at UW-Madison and Mirus Bio

Corporation of Madison, Wis., now reports a critical advance relating to

one of the most fundamental and challenging problems of gene therapy:

how to safely and effectively get therapeutic DNA inside cells.

The Wisconsin scientists have discovered a remarkably simple solution.

They used a system that is virtually the same as administering an IV

(intravenous injection) to inject genes and proteins into the limb veins

of laboratory animals of varying sizes. The genetic material easily

found its way to muscle cells, where it functioned as it should for an

extended period of time.

" I think this is going to change everything relating to gene therapy for

muscle problems and other disorders, " says Jon Wolff, a gene therapy

expert who is a UW Medical School pediatrics and medical genetics

professor based at the Waisman Center. " Our non-viral, vein method is a

clinically viable procedure that lets us safely, effectively and

repeatedly deliver DNA to muscle cells. We hope that the next step will

be a clinical trial in humans. "

Wolff conducted the research with colleagues at Mirus, a biotechnology

company he created to investigate the gene delivery problem. He will be

describing the work on June 3 at the annual meeting of the American

Society for Gene Therapy in Minneapolis, and a report will appear in a

coming issue of Molecular Therapy. The research has exciting near-term

implications for muscle and blood vessel disorders in particular.

Duchenne's muscular dystrophy, for example, is a genetic disease

characterized by a lack of muscle-maintaining protein called dystrophin.

Inserting genes that produce dystrophin into muscle cells could override

the defect, scientists theorize, ensuring that the muscles with the

normal gene would not succumb to wasting. Similarly, the vein technique

can be useful in treating peripheral arterial occlusive disease, often a

complication of diabetes. The disorder results in damaged arteries and,

frequently, the subsequent amputation of toes.

What's more, Wolff says, with refinements the technique has the

potential to be used for liver diseases such as hepatitis, cirrhosis and

PKU (phenylketonuria).

In the experiments, the scientists did not use viruses to carry genes

inside cells, a path many other groups have taken. Instead, they used

" naked " DNA, an approach Wolff has pioneered. Naked DNA poses fewer

immune issues because, unlike viruses, it does not contain a protein

coat (hence the term " naked " ), which means it cannot move freely from

cell to cell and integrate into the chromosome. As a result, naked DNA

does not cause antibody responses or genetic reactions that can render

the procedure harmful.

Researchers rapidly injected " reporter genes " into a vein in laboratory

animals. Under a microscope, these genes brightly indicate gene

expression. A tourniquet high on the leg helped keep the injected

solution from leaving the limb.

" Delivering genes through the vascular system lets us take advantage of

the access blood vessels have - through the capillaries that sprout from

them - to tissue cells, " Wolff says, adding that muscle tissue is rich

with capillaries. Rapid injection forced the solution out of the veins

into capillaries and then muscle tissue.

The injections yielded substantial, stable levels of gene activity

throughout the leg muscles in healthy animals, with minimal side

effects. " We detected gene expression in all leg muscle groups, and the

DNA stayed in muscle cells indefinitely, " notes Wolff.

In addition, the scientists were able to perform multiple injections

without damaging the veins. " The ability to do repeated injections has

important implications for muscle diseases since to cure them, a high

percentage of therapeutic cells must be introduced, " he says.

The researchers also found that they could use the technique to

successfully administer therapeutically important genes and proteins.

When they injected dystrophin into mice that lacked it, the protein

remained in muscle cells for at least six months. Similar lasting power

occurred with the injection of erythropoietin, which stimulates red

blood cell production.

Furthermore, in an ancillary study, the researchers learned that the

technique could be used effectively to introduce molecules that inhibit

- rather than promote - gene expression, a powerful new procedure called

RNA interference.

" This could be very useful if you want to down-regulate a protein that's

causing a muscle disorder, such as with myotonic dystrophy, " says Wolff.

In the late 1980s, Wolff and his UW-Madison colleagues surprised the

scientific world with their discovery that they could get genes to

express in muscle cells simply by injecting naked DNA into rodent

muscle. The Wisconsin Alumni Research Foundation (WARF) licensed the

technology to Vical, a California biotechnology company.

Once Wolff created Mirus, a local company, he and his colleagues turned

their attention to the vascular system, a conduit to multiple leg and

arm muscles they felt would work more efficiently than direct injection

into muscle. WARF licensed the vascular technique to Mirus, which now

holds the patent and continues to commercialize the technique.

In their first studies, the researchers focused on arteries, but then

began to concentrate on veins. " Injecting any substance into arteries

carries a degree of risk since, unlike veins, only one artery feeds a

whole limb, " notes Wolff.

In a related procedure, they experienced excellent results with

high-pressure injection of genes into the tail veins of rodents, a

technique that yielded extensive gene expression in the animals' livers.

" We think the genes traveled from the capillaries through the relatively

large holes that exist in liver cells, " Wolff says, adding that the

technique has become a successful research tool for many laboratories

around the world.

" For delivering genes to limb muscles, the vein approach is so simple, "

he says. " We never expected it to work so well. "

Collaborating on the study were Hagstrom, Hegge, Mark Nobel,

and Hans Herweijer, from Mirus Bio; and Guofeng Zhang and

Vladimir Budker, from the Waisman Center.

http://www.wisc.edu/