By Sean Henahan, Access Excellence

DURHAM, N.C. (June 5, 1996) The successful use of enzymes to to repair faulty genes in living cells could lead to entirely new approaches to gene therapy for diseases such as sickle cell anemia, report researchers at Duke University.

More than a decade ago researchers discovered that instead of being simply a passive carrier of genetic information, the genetic material known as RNA is an active participant in editing genetic messages before they are translated into protein.

The editing approach to gene therapy ignores the defective genes, which are encoded in DNA and stored in the chromosomes, in favor of focusing on the specific genetic RNA messages that are translated into protein. Such messages are copied from the chromosomes into messenger RNA (mRNA). But mRNA copied from DNA is often full of superfluous information that has to be edited out before the mRNA is decoded into the final protein product. Cells have evolved an efficient system that uses RNA enzymes or ribozymes to cut this superfluous data out of mRNA and paste it back together again.

Bruce Sullenger, assistant professor of experimental surgery and genetics at Duke University. . reasoned that ribozymes could be adapted as a tool to recognize defective mRNA and splice in a corrected version. To accomplish this, he turned to the first ribozyme discovered, from the single-celled organism Tetrahymena thermophila. This ribozyme not only cuts other pieces of RNA at specific sites called recognition sequences, but after it cuts, it splices in a piece of RNA sequence attached to its tail end.

A defective gene was introduced into mouse cells growing in a test tube. Then a ribozyme was gentically engineered to recognize a short stretch of RNA near the genetic defect and splice in the corrected sequence, which was produced artificially and attached to the ribozyme tail. When the ribozyme was introduced into the mouse cell, it recognized the defective RNA and swapped in the corrected version

"This research proves that we can use nature's own processes to rewrite genetic instructions in mammalian cells. The results have encouraged us to go forward in exploring the use of this technology to correct disease-causing genetic defects." said Bruce Sullenger, assistant professor of experimental surgery and genetics at Duke University. .

Although the technique is still in the proof-of-concept stage, Sullenger's twist on gene therapy addresses many of the problems that have complicated early gene therapy efforts. Many genetic diseases involve DNA mutations that result in the production of inadequate and or otherwise mutant proteins (eg, sickle-cell anemia or some forms of retinitis pigmentosa). These diseases are considered primary targets for some kind of gene therapy.However, current efforts to replace the defective gene with a new one have not proven effective .

By using ribozymes to introduce corrected genes into the mRNA, the defective gene would remain under the regulatory control of the cell. So when a faulty genetic message is generated, the ribozyme would intercept it and correct it before it is translated into protein.

The ribozyme would also simultaneously decrease the production of faulty protein in the cell and increase the production of functional protein. In traditional gene therapy, functional protein would be added but production of faulty protein could not be stopped.

Sullenger and colleagues have already begun to experiment with correcting the defective gene that causes sickle cell anemia, a disease caused by a single gene defect for making beta globin, part of the oxygen-carrying molecule hemoglobin in blood. This single change causes a red blood cell to be misshapen into the characteristic sickle shape instead of its normal doughnut shape.

Sickle cell anemia would seem to be a prime candidate for gene therapy, because scientists know that a single gene causes the defect. However, production of beta globin is strictly controlled by the cell. Simply adding an additional copy of a good beta globin gene to a cell would probably not cure the disease, Sullenger said, because the defective copy would still be present and the added gene would not be placed under the cell's precise regulatory scheme.

Sullenger has already designed a ribozyme to correct the sickle cell trait. His scheme would allow the gene to stay under the cell's control, but the defective message would be corrected before being translated into protein. Research now underway will test whether this strategy works in mouse cells. If the experiment succeeds, the research team will transfer altered mouse cells to the bone marrow of mice with sickle cell trait to see if they can get stable production of normal hemoglobin in the animals.

Sullenger is also working on a strategy to use ribozymes to alter viral messages. The idea is to use ribozymes to change the meaning of HIV's messenger RNA so that the HIV is tricked into producing an antiviral agent, which would kill the virus when it tries to multiply inside cells. This unique strategy offers the advantage of turning HIV's own genetic messages against themselves. Thus, non-infected cells would not be affected by the ribozymes.

Sullenger stresses that the findings are preliminary and many problems need to be worked out before the strategy could be considered practical. For example, Sullenger discovered that the ribozyme was extremely efficient at seeking out and correcting the defective message. But because the recognition sequence he engineered into the ribozyme is short and appears in many other stretches of RNA in the cell, the ribozyme also targeted some unintended sequences in other RNA molecules. But even though the ribozyme altered a small percentage of unintended RNA sequences, the cells still appeared normal, he said.

"We expected this result. Our next task will be to lengthen the recognition sequence to make it relatively rare, or even unique, in the cell. Other researchers have already shown it is possible to do this, he said.

This research was published in the June issue of Nature Medicine.

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