Gene therapy News

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Stanford Researchers Develop Gene Therapy Technique That Sharply Cuts

 Risks

DATELINE: STANFORD, Calif., Oct. 13, 2002

BODY:

      Researchers at Stanford University Medical Center have

devised a new gene therapy technique that appears to eliminate one of

the major health risks linked to gene therapy. The technique, published

in the Oct. 15 advanced online edition of the journal Nature

Biotechnology, overcomes the need for viral vectors that have plagued

gene therapy trials, while retaining the ability to insert therapeutic

DNA into specific sites in the chromosomes.

       " Our approach provides an alternative that didn't

exist before, " said Michele Calos, Ph.D., associate professor of

genetics at the School of Medicine and lead author on the study.

      The goal of gene therapy is to insert a healthy copy

of a gene into a cell where it can take over for a faulty version. If

the therapeutic DNA does not integrate into the human chromosome, it

produces its protein for a short time before being turned off or broken

down within the cell. For a long-term cure, the gene has to wedge itself

into a chromosome where it remains indefinitely integrated, getting

passed on when the cell divides.

      Current gene therapy approaches that cause genes to

integrate use a viral vector to sneak the therapeutic DNA into the host

cell, Calos said. However, the DNA inserts itself into the chromosome at

random positions. In one recent French gene therapy trial, the randomly

inserted DNA apparently activated a neighboring oncogene, causing a

patient to develop leukemia. " That sort of puts another cloud over the

existing gene therapy trials, " Calos said.

      Calos' technique avoids the pitfalls of other gene

therapy approaches by integrating DNA without using viral vectors,

inserting the DNA at known locations. This new technique can also handle

genes that are too large to fit into a viral package, such as the gene

for Duchenne's muscular dystrophy, Calos said.

      In developing this new approach, Calos hijacked a

mechanism used by a bacteria-infecting virus (called a bacteriophage) to

integrate its genes into bacteria. The bacteriophage makes a protein

called integrase that inserts the viral genes into a specific DNA

sequence on the bacteria chromosome. It turns out that humans also have

a version of that DNA sequence. When the researchers insert a copy of

the therapeutic gene and a gene coding for integrase into a human cell,

the integrase inserts the gene within the human sequence.

      Calos and members of her lab, in collaboration with

Mark Kay, M.D., Ph.D., professor of pediatrics and genetics, tested the

technique using a gene that makes Factor IX--a protein that is missing

in the blood of people with one form of hemophilia. They injected mice

with a piece of DNA containing the Factor IX gene plus a stretch of DNA

that acts as an " insert me " signal to integrase. At the same time they

injected a gene for integrase.

      Within a week, mice that received this injection made

12 times more Factor IX than their littermates that received the

injection without the integrase. Further experiments confirmed that the

Factor IX gene had successfully integrated into the mouse DNA.

      Although the mouse genome contains at least 53

potential integration sites, Calos and her team found the Factor IX gene

in only two locations, with one location by far the more common. She

said that for each tissue there may be a particular site that is the

most likely insertion point. Her group is testing the technique in

different tissue types to ensure that no human integration site is near

a potential oncogene. " We need to look in different tissues to see where

the hot spot is, " Calos said.

      Calos is also modifying the integrase so it targets

specific integration sites that her team knows are safe. " We mutated the

enzyme and evolved it so it will prefer one place over another, " she

said.

      Calos said this approach should be effective for

treating diseases in several different human organs including skin,

retina, blood, muscle and lung. She hopes to start human trials for the

technique in a fatal childhood skin disease called recessive dystrophic

epidermolysis bullosa, which she has already treated in mice. " If that

trial shows that it is safe then that will open the door for trials in

other diseases, " Calos said. She has collaborations underway testing the

technique for use in Duchenne's muscular dystrophy and cystic fibrosis,

among others.

Becki

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