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Aug 22

Rowing upstream: New strategy targets range of toxic effects of ALS gene

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An international team of researchers, including three Packard scientists, have developed a method to interrupt the process that turns the gene into protein

Work from yeast, flies, worms, and cultured neurons from patients reveal a novel way to target the most common genetic cause of amyotrophic lateral sclerosis (ALS). Instead of directly targeting the genetic mutation in the C9orf72 gene, an international team of researchers, including Packard scientists Aaron Gitler of Stanford University, Fen-Biao Gao of the University of Massachusetts Medical School, and Leonard Petrucelli of the Mayo Clinic, developed a method to interrupt the process that turns the gene into protein. The work, published in Science, introduces a new strategy to potentially slow the progress of ALS.

“It’s a basic biology question with an ALS application. The protein we targeted specifically reduces the transcription of long repeats, but not other genes, including the normal version of C9orf72,” said Nicholas Kramer, first author of the Science paper.

The genetic mutation in the C9orf72 gene is known as a repeat expansion, in which six nucleotides (GGGGCC) are repeated hundreds, even thousands, of times. Not only does this potentially alter the normal function of the gene, it also creates its own toxic byproducts. The large number of repeats confuses the machinery that turns DNA into RNA, and then into protein, causing it to transcribe the repeat expansion both forwards and backwards (known as sense and antisense RNA). As a result, the cell produces large amounts of short RNAs and small proteins called dipeptide repeats, both of which have previously been shown to harm the cell.

The authors realized that directly blocking the effects of the C9orf72 repeat expansion would require interfering with both the forwards and backwards RNA transcripts, since both generate harmful byproducts. Several recent studies on Huntington’s disease, which is also caused by a repeat expansion, provided an answer. Researchers were able to reduce some of the harmful effects of the repeat expansion by blocking a type of protein called a transcription elongation factor. These proteins help the cell build a long strand of RNA using the DNA of a gene as a template. Interfering with a specific transcription elongation factor called Spt4 in Huntington’s disease prevented the synthesis of the toxic protein from the repeat expansion. Just as importantly, it didn’t affect the production of the rest of the proteins the cell needed to function.

When the team led by Gitler and Petrucelli deleted the Spt4 gene in yeast that were engineered to carry a copy of the human C9orf72 gene with 66 repeats, they found a significant reduction in the specific types of damaging RNA and small proteins associated with the repeat expansion. Other experiments revealed that deleting Spt4 didn’t have any negative effects on other yeast genes, which is important if blocking Spt4 is to be a viable clinical target in humans.

Next, the researchers moved to models in Caenorhabditis elegans worms and Drosophila melanogaster fruit flies. Reducing the amount of Spt4 produced in the worms not only decreased the amount of deleterious RNAs and dipeptide repeat proteins, it also increased the worms’ survival. The experiments in fruit flies had similar outcomes, indicating that blocking or reducing the activity of Spt4 could reduce the pathogenicity of the C9orf72 repeat expansion.

Gitler, Petrucelli, and colleagues also tested their hypothesis in cultured fibroblasts from humans with C9orf72 ALS. When they blocked the human version of Spt4 RNA with specially designed interfering RNAs, the cells had lower amounts of both toxic RNA and proteins that were produced from the forwards and backwards reading of the repeat expansion DNA. Blocking this RNA only affected a small subset of cellular genes, which makes it more likely that it will be safe in animal and human tests.

Researchers are still a long way off from actually turning these discoveries into ALS treatments, but the work provides a novel target to investigate, Kramer says.

“It seems to make more sense to find something more upstream, rather than develop two separate drugs targeting both sense and antisense gene products,” Kramer said.

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