Silencing of mutant genes with RNAi.

RNA works hard at the business of expressing genetic information. It carries instructions from DNA in the cell nucleus into the cytoplasm, where basic housekeeping functions are carried out and proteins manufactured. When messenger RNA arrives in the cytoplasm, it binds to the ribosomes and guides the assembly of amino acids into proteins. Now advances in genomics have led to the discovery that, in addition to its transport and manufacturing roles, RNA can silence gene expression by a process called RNA interference (RNAi). RNAi provides a new tool for investigating gene function that also has potential for developing novel clinical treatments for certain previously untreatable diseases.

RNAi is an evolutionarily conserved cellular mechanism in worms, plants, and animals. In the RNAi pathway, long pieces of double-stranded RNA are cut into smaller pieces by the "dicer" enzyme to form small interfering RNAs (siRNAs) that are about 21 nucleotides long. These siRNAs bind with other molecules to form the RNA-induced silencing complex, which allows the siRNAs to target specific messenger RNAs to block production of protein.

In the 10 June 2003 issue of Proceedings of the National Academy of Sciences, graduate research assistant Victor M. Miller, associate neurology professor Henry L. Paulson, and colleagues at the University of Iowa report results that will faciliate development of siRNA therapies for heritable diseases such as Machado-Joseph disease (MJD) and other dementias in which defective proteins clump together and impair brain and nervous system function. In MJD, for example, a mutation of the MJD1 gene produces multiple copies of the amino acid glutamine, which makes a protein that is toxic to cells. And in frontotemporal dementia with parkinsonism, a mutation of the tau gene produces defective tau protein consisting of the tangled filaments that lead to cell death in dementia disorders such as Alzheimer disease.

Miller and colleagues conducted experiments using siRNAs to silence genes of these two diseases. In the experiments, siRNAs were produced in a test tube, then added to cells to see if they inhibited or suppressed expression of the targeted gene. Sequences for siRNAs that worked were inserted into a plasmid for production of short hairpin RNA, which the cell converts into siRNA, using the specific sequence for each different siRNA the team wanted to clone.

Targeting a single-nucleotide difference between the mutant and healthy MJD1 gene enabled the scientists to almost completely eliminate production of the defective protein in a human cell model system. The experiments using siRNAs to knock down expression of the tau gene also succeeded in reducing production of the protein that causes disease.

Paulson says, "Because the human genome is full of polymorphisms, including countless single-nucleotide polymorphisms, it is conceivable that some of these might be associated with diseases, or traits, that allow them to be the 'hook' by which selective targeting [of a gene] can occur." The ability of siRNAs to knock down disease-causing proteins coded by dominant genes offers hope for new and effective treatment of diseases that other genetic engineering strategies--such as gene replacement therapy--can not address.

According to Hui Zhang, an associate professor of genetics at the Yale University School of Medicine, the paper is highly significant in that it provides a conceptual as well as potential way of treating diseases that contain point mutations through interfering with normal cellular function. "The authors provided detailed analysis for allele-specific silencing of the disease genes using the siRNA approach, which may provide a therapeutic answer to many mutation-based diseases," says Zhang. "Their conclusion ... is consistent with our understanding of the siRNA targeting mechanism reported by many others."

The next big hurdle will be to test siRNAs in an animal model. In a collaboration between Paulson and Beverly Davidson, the Roy J. Carver Associate Professor of Internal Medicine at the University of Iowa, the team is now employing a viral vector to introduce siRNAs into mouse models of human neurodegenerative disease.

Although human therapy is the ultimate goal, there are a number of challenges ahead before this new technology will be available. These include possible rapid degradation of siRNA in the cell, nonspecific effects on gene expression, and the need for high specificity to prevent unwanted side effects of treatment, such as possible interference with other proteins or biological pathways, or elicitation of immune system responses.