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Duke Researchers Show "Editing" Can Fix Faulty Genes in New Approach to Gene Therapy

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Duke Health News 919-660-1306

DURHAM, N.C. -- Duke University Medical Center researchers have shown for the first time that enzymes can be used inside living cells to repair faulty genetic messages, instead of replacing them.

The finding, published in the June issue of Nature Medicine, opens up a new realm of possibilities for correcting genetic information, according to Bruce Sullenger, assistant professor of experimental surgery and genetics. The Duke team already has begun exploring use of these new therapeutic approaches for sickle cell anemia and even to "sabotage" the AIDS virus.

"This research proves that we can use nature's own processes to rewrite genetic instructions in mammalian cells," Sullenger said in an interview. "The results have encouraged us to go forward in exploring the use of this technology to correct disease-causing genetic defects."

The study was funded by a grant from the National Institutes of Health. Duke researchers Joshua Jones and Seong-Wook Lee also contributed to the work.

Sullenger's strategy is based on the discovery, more than a decade ago, 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 a portable form called 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 junk out of mRNA and paste it back together again.

Sullenger 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.

To test his idea of repairing faulty genes, Sullenger introduced a defective gene into mouse cells growing in a test tube. Then he engineered a ribozyme to recognize a short stretch of RNA near the genetic defect and splice in the corrected sequence, which Sullenger had produced artificially and attached to the ribozyme tail. When he introduced the ribozyme into the mouse cell, it recognized the defective RNA and swapped in the corrected version.

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.

In traditional gene therapy, the newly introduced gene cannot be placed under the cell's normal regulatory controls -- stretches of genetic code that help control when the gene should be turned on or off -- because the viruses that carry new genes into cells are too small to include the control codes. 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 already has begun to experiment with correcting the defective gene that causes sickle cell anemia, a disease caused by a single "misspelling" in the gene 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. The result is a devastating disease that affects thousands of people, mostly African-Americans, in the United States. Researchers estimate that one in 10 African-Americans carries a copy of the defective sickle cell gene.

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 in Sullenger's lab will test whether this strategy works in mouse cells. If he 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.

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