A New Method to Model Diseases from Splicing Defects
Scientists have 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
Targeting tiny snippets of RNA called ASOs (antisense oligonucleotides), scientists at Cold Spring Harbor Laboratory (CSHL) have found 'negative ASOs' cause missplicing and pathogenesis, providing unique windows on disease progression.
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 new treatments. It was tested successfully 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 today in Genes & Development.
The modeling method is called TSUNAMI (shorthand for targeting-splicing using negative ASOs to model illness).
The new study demonstrates it can be used
in illnesses with pathologies associated
with the missplicing of pre-mRNAs
unedited RNA molecules carrying messages
encoded in genes providing instructions
for how 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 cell factories where proteins are synthesized.
In SMA and some other human illnesses, pathology can be traced to errors in the pre-mRNA editing process.
SMA, 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.
The SMN protein normally encoded by the gene
is essential for motor neuron development.
Humans have a second, similar gene called SMN2,
but it's 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 the 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 which results in pathology of varying intensity, Krainer - a leading expert on splicing - and his collaborators have recently succeeded in devising a method to get SMN2 to produce therapeutic amounts of the protein, enough to reverse pathology in both mild and severe mouse models of the disorder.
They did this by synthesizing tiny snippets of RNA called ASOs (antisense oligonucleotides) and injectingd them into the cerebrospinal fluid of mice carrying a human SMN2 transgene (a gene not native to mice). This enabled the therapeutic ASOs to get through the blood-brain barrier and reach cells throughout the central nervous system.
ASOs are configured to attach at
specific locations on pre-mRNAs
where they inhibit activators or repressors
of the splicing process.
Krainer's team was able to synthesize an ASO that corrected the SMN2 splicing error and gave rise to therapeutic amounts of the SMN protein. Importantly, that ASO was shown to be stable in the body as well as be persistent, so that the effects of a single injection lasted at least half a year in mice.
A version of this therapeutic ASO is now being tested in Phase 1 human trials.
TSUNAMI's 'negative ASOs': therapy in reverse
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. An approach they call TSUNAMI.
The team created ASOs that target another unique site on the pre-mRNA of the human SMN2 gene. When these negative ASOs were injected into the ventricular brain of mice (engineered to have four copies of the SMN2 transgene, and in addition lack the mouse's own Smn gene) SMA symptoms became more severe than in control (normal) animals.
With four copies of the transgene, untreated mice only have very subtle symptoms. But when the negative ASOs were injected immediately following birth, severe SMA pathology developed according to dose, and in a progressive fashion. The team then "rescued" these mice by injecting them with the therapeutic ASO they had previously shown can correct the SNM2 splicing error.
Krainer: "This amounts to a proof of principle. By using TSUNAMI we are able to dissect pathogenesis mechanisms, including spatial and temporal features of disease onset and progression. We are also able to observe the impact of candidate therapeutics in the same manner, both as a function of where they have their impacts in the body and when."
TSUNAMI will provide new modeling alternatives
Kentaro Sahashi, a postdoctoral fellow who is first author on the new paper and a member of the Krainer lab, stresses that TSUNAMI is offered as an alternative and complement to other methods of making animal models of human illness.
By knocking out genes, or modifying their expression patterns, molecular biologists have made great strides in understanding human cancers. Sahashi: "These are very powerful technologies, and TSUNAMI does not in any way replace them. Rather, we think of it as providing specific advantages in modeling certain illnesses."
TSUNAMI has the advantage, in splicing-related illnesses, of inducing pathology by precisely mimicking mechanisms that give rise to illness in people. It can also help identify clinical biomarkers for illnesses.
Krainer: "You can generate a disease and rescue it; you can generate a series of mice treated in different ways. And using mass spectrometry and other techniques, you can look for biomarkers. Then, of course, you have to test to see if these putative markers are clinically relevant. This is a powerful technology for doing this sort of work, and not only in SMA."
Krainer further notes that negative ASOs can be used to target pre-mRNAs of genes that are naturally occurring in an animal rather than of a human transgene, the target in the experiments reported today. This way, he says, "one should be able in principle to generate splicing-associated disease models in virtually any wild-type species."
Krainer's lab at CSHL may soon turn to another illness, familial dysautonomia, caused by a splicing error. But splicing errors also crop up in many other illnesses, from cystic fibrosis to cancer.
Krainer: "We can't possibly fix every splicing defect found in these diseases, but we can certainly cause something analogous to them and learn from the models we make."
"TSUNAMI: an Antisense Method to Phenocopy Splicing-Associated Diseases in Animals" appears online ahead of print in Genes & Development August 14, 2012. The authors are: Kentaro Sahashi, Yimin Hua, Karen K.Y. Ling, Gene Hung, Frank Rigo, Guy Horev, Masahisa Katsuno, Gen Sobue, Chien-Ping Ko, C. Frank Bennett and Adrian R. Krainer. The paper can be obtained online at: http://genesdev.cshlp.org/
This research was supported by grants from National Institutes of Health (Grant 2R37GM042699-22], the Muscular Dystrophy Association, the SMA Foundation, and the St. Giles Foundation.
Founded in 1890, Cold Spring Harbor Laboratory (CSHL) has shaped contemporary biomedical research and education with programs in cancer, neuroscience, plant biology and quantitative biology. CSHL is ranked number one in the world by Thomson Reuters for impact of its research in molecular biology and genetics. The Laboratory has been home to eight Nobel Prize winners. Today, CSHL's multidisciplinary scientific community is more than 360 scientists strong and its Meetings & Courses program hosts more than 12,500 scientists from around the world each year to its Long Island campus and its China center. Tens of thousands more benefit from the research, reviews, and ideas published in journals and books distributed internationally by CSHL Press. The Laboratory's education arm also includes a graduate school and programs for undergraduates as well as middle and high school students and teachers. CSHL is a private, not-for-profit institution on the north shore of Long Island. For more information, visit www.cshl.edu.
Original article: http://www.eurekalert.org/pub_releases/2012-08/cshl-cti081012.php