Designer molecules correct RNA splicing defects: New strategy for treating many diseases

With a high-tech fix for faulty cellular editing, scientists at Cold Spring Harbor Laboratory have moved a step closer to developing treatments for a host of diseases as diverse as breast cancer, muscular dystrophy, and cystic fibrosis.

Many human diseases have been linked to defects in a cellular editing process called pre-messenger RNA splicing. Adrian Krainer, a molecular biologist at Cold Spring Harbor Laboratory, has spent years investigating this complex editing process, which takes the information coded in genes and makes it available for building proteins. In a new study published in the journal Nature Structural Biology, Krainer’s team has devised a clever way to correct RNA splicing defects implicated in breast cancer and spinal muscular atrophy (a neurodegenerative disease). In principle, the technique could provide the ability to correct RNA splicing defects associated with any gene or disease.

For now, Krainer’s method has been shown to work under the simplest of conditions — in test tubes with small segments of RNA. The next step is to adapt the technique for use in living cells. Still, “It’s a very promising approach,” says molecular biologist Brenton Graveley, of the University of Connecticut Health Center. “There are a lot of hurdles to be overcome in terms of delivering the corrective molecules to the cells that need to be treated. But theoretically the exact same approach could be taken for any gene at all, and the list of genes that have defects at the level of RNA splicing is very long,” says Graveley, who is familiar with the research but not involved in the study.

For cells to produce protein, DNA is first transcribed into pre-messenger RNA (RNA is a chemical cousin of DNA). Pre-messenger RNA is a “word-for-word” representation of a DNA sequence in the language of RNA. But for reasons that remain unclear to scientists, pre-messenger RNA molecules contain excess “words” that are removed by splicing to create mature messenger RNA (mRNA), the templates that cells use to make proteins. In many genetic diseases, gene mutations cause errors in the RNA splicing process. Improperly spliced mRNA molecules lead to the creation of altered proteins that cannot perform their duties properly, resulting in disease.

Gene mutations that alter pre-mRNA splicing frequently cause an important segment of the RNA to be skipped or left out of the mature mRNA. With this in mind, Krainer and colleague Luca Cartegni looked for ways to tell a cell to include a piece of RNA that is erroneously skipped. They took inspiration from natural proteins that guide which segments are included when the cell’s splicing machinery cuts up pre-mRNA and pastes only the important bits back together. One end of these guide proteins attaches to the pre-mRNA transcript. The other end recruits enzymes that carry out the actual cutting and pasting.

Krainer and Cartegni attached the recruiting portion of the guide protein to a synthetic molecule that can be programmed to bind to any piece of RNA according to its sequence. The researchers designed a batch of these molecules corresponding to a mutant form of the BRCA1 gene implicated in breast cancer. The designer molecules successfully caused the splicing machinery to include an important piece of BRCA1 mRNA that is usually skipped. Thus, the designer molecules corrected the splicing error, making a normal messenger RNA from a defective pre-messenger RNA transcript.

Next, the scientists turned their new technology loose on a mutant form of the SMN2 gene which is associated with the neurodegenerative disease spinal muscular atrophy (SMA). People afflicted with SMA generally possess both a fully defective SMN1 gene and one or more copies of the closely related SMN2 gene which, due to skipping of a particular segment during RNA splicing, is capable of producing only small amounts of normal mRNA. The severity of SMA symptoms could be relieved if a patient’s SMN2 gene could be coaxed into producing more normal mRNA by including the skipped RNA segment more often. Just as they corrected splicing defects of BRCA1 RNA, Krainer and Cartegni’s designer molecules also enhanced the production of properly spliced SMN2 RNA.

The scientists dubbed the method ESSENCE (which stands for Exon-Specific Splicing Enhancement by small Chimeric Effectors). The next step is to create ESSENCE designer splicing molecules that pass easily into cells and can home-in on the desired splicing targets. The new study establishes that if such molecules can be developed, they may ultimately prove useful for treating a great diversity of human disease.