In addition to silencing the mutated gene that causes ALS, the EPFL researchers were able to simultaneously deliver a normal version of the gene to motor neuron cells using a single delivery mechanism. "This is the first proof of principle in the human form of a disease of the nervous system in which you can silence the gene and at the same time produce another normal form of the protein," notes Patrick Aebischer, EPFL President and a co-author of the study.
ALS is a progressive neurological disease that attacks the motor neurons controlling muscles. Although its victims retain all their mental faculties, they experience gradual paralysis and eventually lose all motor function, becoming unable to speak, swallow or breathe. Known also as Lou Gehrig's disease, from the baseball player who succumbed to it, this harrowing disease has no cure and its pathogenesis is not very well understood.
An estimated 5,000 Americans are diagnosed with ALS every year, and most of these cases are "sporadic", with no identifiable cause. About 5-10% of ALS cases are inherited. Of these, 20% have been linked to any of more than 100 mutations in the gene that expresses the superoxide dismutase enzyme (SOD1).
These SOD1 mutations are "toxic gain-of-function mutations," meaning that the protein expressed by the mutated gene has, in addition to all its normal cellular functions, some additional function that makes it toxic to the cell. "Any mutation to the SOD1 gene is fatal to motor neuron cells," Aebischer notes. Recent research also indicates that mutant SOD1 gene expression in neighboring glial cells is also implicated in motor neuron death.
Lead author Cedric Raoul and colleagues targeted the cause of the disease by using RNA interference to silence the defective gene, preventing it from expressing the SOD1 protein.
RNA interference is part of a complex cellular housekeeping process that protects cells from invading viruses or other genetic threats. It works by interrupting messenger RNA as it transfers the genetic code for a protein from the nucleus to the site in the cell where the protein is synthesized.
To trigger RNA interference and silence a gene, short bits of double-stranded RNA are introduced in the cell, where they bind with matching sections of messenger RNA. The cell identifies the resulting messenger RNA strand as faulty and chops it up. As a result, the genetic blueprint isn't delivered and the protein never gets made.
"Gene silencing is an example of using "molecular scissors" at its most advanced level," Raoul explains.
Raoul and colleagues used RNA interference to reduce levels of mutant SOD1 protein in the spinal cords of transgenic ALS mice (mice bred to express the human SOD1 gene). Short strands of RNA that targeted multiple mutated and normal forms of the human SOD1 gene were delivered in a specially engineered lentivirus. Expression of the SOD1 protein was knocked down in the affected motor neurons and neighboring glial cells, and both the onset and the rate of progression of the disease in the treated mice were substantially reduced. In addition, the mice showed a significant improvement in neuromuscular function.
"This is the first demonstration of therapeutic efficacy in vivo of RNA interference-mediated gene silencing in an ALS model," notes Raoul.
Because the normal form of the SOD1 protein may be necessary for the survival or function of adult human motor neurons, the Swiss researchers designed a gene replacement technology that allows the knock-down of all mutant SOD1 forms while permitting the expression of a normal type SOD1 protein that is resistant to RNA interference-based silencing. Both these effects are expressed long-term via delivery by a single lentiviral vector.
Aebischer is optimistic about the future of gene silencing as a potential therapy, particularly in incurable progressive neurological diseases such as ALS. "I would not be surprised to see, in the next ten years, this technology used for treating diseases of the nervous system, particularly diseases that involve toxic gain-of-function, such as inherited forms of Parkinson's disease or Huntington's disease," notes Aebischer. "But it's important to note that the safety of delivering lentiviral vectors to the nervous system will have to be carefully examined prior to treating patients."
This research was supported in part by The ALS Association (ALSA) and the Swiss National Science Foundation.