June 11, 2003
Selective Blocking Of Mutant Genes Demonstrated

University of Iowa graduate student Victor Miller and other researchers have demonstrated the ability to selectively turn off a copy of a gene that differs by only a single nucleotide from other copies of the same gene in the same cell.

Turning off a mutant gene while keeping the normal gene active would be particularly useful in therapies aimed at treating so-called dominantly inherited diseases. In these diseases, a single mutant copy of a gene inherited from either parent dominates the normal gene by producing a protein that is toxic to cells. Thus, a successful therapy must remove or suppress the disease-gene rather than simply add a corrected version. At the same time, the normal gene may be essential, so it is important to be able to silence the disease-causing gene without affecting the normal copy. Many neurodegenerative conditions, including Huntington's disease (HD), are dominantly inherited. The HD gene also is an example of a normal gene that appears to be essential for normal function.

Working in cell culture, the UI researchers used the relatively new technology known as RNA interference to silence a mutant gene that causes the neurodegenerative condition called Machado-Joseph disease (or Spinocerebellar Ataxia Type 3), while leaving the normal gene alone.

Machado-Joseph disease (MJD), Huntington's disease and at least seven other neurodegenerative disorders all are caused by the same type of genetic mutation. The genetic defect in these diseases produces a mutated protein with an abnormally long stretch of a repeated amino acid. The mutant protein in each of these conditions is prone to clump together, forming aggregates, which appear to damage brain tissue. Other neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, also are characterized by a tendency for proteins to misfold or clump in the brain. The UI team studies Machado-Joseph disease because it is a good model for investigating these types of neurodegenerative diseases.

Initial attempts to silence the mutant MJD gene by targeting the RNA interference to the repeat-expansion mutation failed. So the UI researchers focused on a single sequence difference, also known as a single nucleotide polymorphism (SNP), which occurs just next to the mutated sequence in about 70 percent of mutant MJD genes.

"When we tried to target the mutation itself, the interfering RNA was not able to distinguish the mutant gene from the normal gene and both copies were suppressed," said Henry Paulson, M.D., Ph.D., UI assistant professor of neurology and principle investigator of the study. "Then we noticed that there was a single nucleotide polymorphism in the mutant MJD gene that comes right after the mutation in most cases. We targeted that single nucleotide variation with RNA interference and that approach was able to distinguish the mutant from the normal and only knock down the mutant gene."

Paulson added that the discovery that RNA interference could distinguish between genes on the basis of a single nucleotide polymorphism was very exciting because every person's DNA differs mostly on the basis of these unique single letter variations in the genetic code. Thus it might be possible to use RNA interference to target unique single nucleotide polymorphisms associated with specific genes in order to manipulate those genes.

"Even when one cannot target a disease-causing mutation, it may still be possible to target the mutant gene on the basis of a SNP associated with that gene," Paulson said.

The research team also used RNA interference to target an actual disease-causing mutation due to a single base pair change in a gene. Tau is an important cellular protein that is mutated in some inherited dementias that are somewhat similar to Alzheimer's disease. The UI researchers directed RNA interference against a specific mutation in the Tau gene that is known to cause dementia in people. Again, the approach was successful in silencing only the mutant gene and not the normal gene.

This result is important because it is harder to replace a gene in a cell than it is to add another gene. Picture a cell that has two different variants of the same gene. Most cells have at least 2 copies of almost every gene (a notable exception being the genes that are on the X chromosome in males). Some people have genetic diseases that are the result of a dominant mutation. When a harmful mutation is dominant only one of the two copies of a gene has to have the mutation in order for the mutation to have harmful effects. The ability to effectively turn off the expression of just the harmful copy would be very valuable. This report provides evidence that even a point mutation of a single nucleotide in the DNA sequence of a gene (called a Single Nucleotide Polymorphism or SNP) provides enough of a difference to be targetted by RNA interference therapies.

To make RNA interference useful for many genetic disorders what is needed is a gene therapy that will cause copies of the interfering RNA sequence to be present in cells for a long time. To do that what is needed is the ability to add DNA to a cell that would persist and continually be used to make interfering RNA. The big enabling technologies needed are mini-chromosomes and a mechanism for delivering mini-chromsomes into large numbers of cells of a target cell type (e.g. into all the neurons in a brain). If a very small mini-chromosome could be added to a cell that expressed a sequence that could do RNA interference against the harmful variation of a gene then it would be possible to prevent the harmful gene from causing problems.

Share |      Randall Parker, 2003 June 11 03:01 PM  Biotech Manipulations

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