LONDON - Geneticists may one day be able to harness the power of naturally occurring enzymes to treat inherited diseases caused by the abnormal repetition of a nucleotide triplet.
Researchers at the University of Oxford, in the U.K., have shown that RNA enzymes called ribozymes can be modified so they reduce the number of repeated nucleotide triplets in RNA and can carry out this procedure in human cells.
Several diseases, including Huntington's disease, have recently been shown to be associated with abnormal numbers of repeated nucleotide triplets, which are known as trinucleotide repeat expansions (TREs).
In myotonic dystrophy (DM), for example, there is a TRE in part of the gene for the enzyme myotonic dystrophy protein kinase (DMPK). Some evidence suggests that the symptoms of this disease may be due to altered amounts of DMPK, which plays a role in cell signaling.
DM is the most common inherited neuromuscular disease, affecting about one in 8,000 adults. The disorder, which is autosomal dominant, causes myotonia, as well as muscle weakness and wasting.
Normal individuals have between five and 35 repeats of the triplet CTG in their DMPK gene, but those affected by DM have between 50 and 2,000 such repeats. The greater the number of repeats, the greater the severity of the disease.
Leonidas Phylactou, research fellow in the department of human anatomy at the University of Oxford, and colleagues have now shown that it is possible to delete the excessive number of repeats from the RNA containing the message for DMPK, using ribozymes. They reported their results in the April 1998 issue of Nature Genetics, in a letter titled “Ribozyme-mediated trans-splicing of a trinucleotide repeat.“
Ribozymes are RNA enzymes. They occur naturally, catalyzing reactions such as cutting RNA, or deleting sections of RNA and then rejoining the cut ends. They normally carry out these reactions on themselves, but it is possible to engineer them to catalyze the same reactions on other RNA molecules.
Phylactou and his colleagues, Charlotte Darrah and Matthew Wood, therefore constructed a “group I intron ribozyme“ specific for a sequence upstream of the region of the DMPK gene which contains the repeated trinucleotides. This ribozyme carried a new section of DMPK mRNA containing only five copies of the repeated trinucleotides.
After confirming that this enzyme could modify DMPK mRNA containing 12 trinucleotide repeat units and replacing these with the new section of mRNA containing only five repeats, the researchers went on to find out whether they could encourage this reaction in mammalian cells.
They transfected the genetic sequence for the ribozyme into human skin fibroblasts. These cells express DMPK, and Phylactou and his colleagues were able to show that the ribozyme could indeed replace the naturally occurring repeated region with that attached to the ribozyme.
Writing in Nature Genetics, the team concluded, “This study represents a step towards exploiting ribozymes for the repair of a mutated endogenous transcript that contains an expanded triplet repeat implicated in human disease.“
Evaluation In Animal Models Among Next Steps
The type of ribozyme used, the researchers added, may have the potential to repair targeted transcripts associated with a broad spectrum of inherited diseases.
Phylactou told Bioworld International, “This is the first example of this kind of ribozyme being used to repair endogenous RNA. The only evidence before this study was with bacterial RNA, but we have now shown that this ribozyme can do the reaction on naturally occurring RNA, and on an RNA which is involved with this disease.“
Theoretically, he added, any RNA could be repaired by this method. “Even if you have a point mutation, it should be possible to replace the mutation with the normal sequence,“ he said.
The group's next target is to try to repair mRNA for DMPK which has very large TREs. “This will be more challenging,“ said Phylactou, “because mutant DMPK mRNAs, which conform to a different structure, may limit access to the ribozyme.“
In the long term, the group expects to be able to evaluate the method in an animal model. Although there is no good animal model at present, Phylactou expects that a transgenic model for DM may exist by the time he and his colleagues are ready to test their approach.
He said: “We would like to be able to show that, by replacing the number of repeats with a normal number, you can actually modify some of the symptoms.“
The group is interested in collaborating with a biotechnology company in order to develop its work further.
As with all potential approaches to gene therapy, however, the success of the scientists' method will depend on whether it can readily be delivered to the cells and tissues that need the corrected genetic material in order to function in a normal fashion. *