ScienceDaily (Aug. 14, 2012) A team led by scientists at Cold Spring Harbor Laboratory (CSHL) has developed a new way of making animal models for a broad class of human genetic diseases -- those with pathology caused by errors in the splicing of RNA messages copied from genes. To date, about 6,000 such RNA "editing" errors have been found in various human illnesses, ranging from neurodegenerative disorders to cancer.

The new modeling approach can provide unique insights into how certain diseases progress and is likely to boost efforts to develop novel treatments. It was tested successfully by the CSHL team, led by Professor Adrian Krainer, Ph.D., in collaboration with scientists from Isis Pharmaceuticals, in mouse analogs of human spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of childhood mortality. The results are detailed in a study published August 15 in Genes & Development.

The modeling method is called TSUNAMI (shorthand for targeting-splicing using negative ASOs to model illness). The study demonstrates it can be used in illnesses with pathology associated with the missplicing of pre-mRNAs -- unedited RNA molecules that bear the messages encoded in genes which provide instructions for cells to manufacture specific proteins.

Correcting splicing errors in SMA

A cellular machine called the spliceosome routinely snips non-essential bits called "introns" out of every pre-mRNA molecule that carries a copy of a gene's instructions. All that should remain after the spliceosome has done its work is a string of spliced-together "exons," the protein-encoding portions of the message. These edited mRNA messages are subsequently read by ribosomes, the cellular factories where proteins are synthesized.

In SMA and some other human illnesses, pathology can be traced to errors in the pre-mRNA editing process. In SMA, it is caused either by a severe mutation in a gene called SMN1 ("survival of motor neuron-1") or by that gene's complete absence in an affected individual's genetic material. The SMN protein normally encoded by the gene is essential for motor neuron development. Humans have a second, similar gene called SMN2, but it is a poor backup. Because of an error in the splicing of its pre-mRNA, the SMN2 gene, when expressed, typically produces only a fraction of the SMN protein needed by motor neurons. This is critical in the first stages of life when the body and muscles are still developing.

While the level of the "backup" gene's protein output varies in individuals with spinal muscular atrophy, resulting in pathology of varying intensity, Krainer -- a leading expert on splicing -- and his collaborators have succeeded in recent years in devising a method of getting SMN2 to produce therapeutic amounts of protein, enough to reverse pathology in both mild and severe mouse analogs of the disorder.

To achieve this they synthesized tiny snippets of RNA called ASOs (antisense oligonucleotides) and injected them into the cerebrospinal fluid of mice carrying a human SMN2 transgene (i.e., a gene not native to mice). This enabled them to get the therapeutic ASOs through the so-called blood-brain barrier, to reach cells throughout the central nervous system. ASOs are configured to attach at highly specific spots in pre-mRNAs, where, by design, they can inhibit activators or repressors of the splicing process. Krainer's team synthesized an ASO that corrected the SMN2 splicing error and gave rise to therapeutic amounts of SMN protein. Importantly, the ASO was shown to be stable in the body as well as persistent, the effects of a single injection lasting at least half a year in mice.

TSUNAMI's 'negative ASOs': therapy in reverse

A version of this therapeutic ASO is now being tested in Phase 1 human trials. But even as the tests proceed, Krainer and colleagues have worked on getting the splice-correction method to work in reverse: using a "negative ASO" to cause or exacerbate disease pathology in neonatal mice, with the approach they call TSUNAMI.

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New method to closely model diseases caused by splicing defects