With the mutation of the alpha-synuclein gene having been identified as a key contributor to the development of familial Parkinson's disease, a number of researchers see the disorder as an ideal one for RNAi intervention.
Among these is a group at Northwestern University, which thinks it may have developed an effective RNAi-based treatment for the disease and hopes that forthcoming in vivo data may lead to a collaboration with industry and ultimately human trials.
Earlier this year, Martha Bohn and colleagues at Northwestern published data in Experimental Neurology demonstrating that an shRNA-expressing lentiviral vector could be used to silence both endogenous human alpha-synuclein in the human dopaminergic cell line SH-SY5Y and experimentally expressed human alpha-synuclein in vivo in the rat brain.
According to Mohan Sapru, an assistant professor in Bohn's lab and co-author of the Experimental Neurology paper, the biggest challenge was designing an shRNA that would down-regulate the mutant form of the alpha-synuclein gene while leaving the wild-type unaffected.
"We have achieved for the first time selectivity and precision [that allows] us to turn off the mutant form of the gene without affecting the wild-type gene," he told RNAi News this week, noting that all the work was done in-house.
Sapru said that the Bohn team has now begun developing the rat models necessary to test the shRNA in an adeno-associated viral vector, which he expects would be used in any human studies.
"We have achieved for the first time selectivity and precision [that allows] us to turn off the mutant form of the gene without affecting the wild-type gene."
The work with lentiviral vectors helped establish proof of concept, and "will also be useful if you want to do something in vitro," he said. "But as far as a clinical trial is concerned, we will use [an] AAV [vector] as our first choice" since they are believed to be safer and are already being tested in US Food and Drug Administration-approved clinical trials.
Though this effort is underway, "the problem is … you have to wait on some of the models for eight months to nine months," Sapru said. Once the mutant alpha-synuclein gene is administered into rodents, the cell death and behavioral abnormalities that are the hallmarks of Parkinson's disease aren't seen for months. Although this is a positive from the standpoint that it mimics the real-life gradual progression of the disease, it is time-consuming.
Once the animal models are ready, the researchers use stereotaxic injections to deliver the shRNA-bearing vectors into the substantia nigra and striatum brain regions, which are the areas primarily affected by Parkinson's disease.
"The advantage of this approach is that you could go to a particular region [of the brain] and do whatever you wanted to do in terms of expressing a gene or turning off a gene, and not letting the rest of the brain cells be affected," he said, adding that this kind of delivery method would also be used in human studies of the RNAi therapy.
Although administration of a drug directly to the brain is a complicated and expensive process, Sapru sees the use of expressed shRNAs as key to making the therapy viable.
"There are two ways to silence a gene [with RNAi]: One would be to make siRNAs … synthetically, but the problem is that they last only about three to four days and then they get degraded," he said. He added that even modified siRNAs are relatively short-lived, making them impractical for a disease like Parkinson's where the neurodegeneration is gradual.
The other way is to use expressed RNAi, which could use a single administration to silence a target gene for a long period. "In the field of gene therapy, at least in animals, people have reported the expression of lentivirus-delivered genes for months to years," Sapru said.
Should the Parkinson's disease therapy being developed by Sapru and his colleagues prove effective, he said that he envisions it being used in conjunction with a genetic test to identify patients with the mutant alpha-synuclein gene.
"In the future, if a patient knows with genetic testing that he or she has the mutation … then one could approach this in a preventative fashion and not have the disease develop in the first place," he said. Another scenario is one in which the therapy is used as a rescue approach in patients who already have some neurodegeneration.
"Even if [disease] onset has taken place … there is the possibility [of] only 5 or 10 percent loss of dopamine neurons in the patient," Sapru said. "It has been shown that [sufferers] have to lose at least 80 to 90 percent of these dopamine neurons before [they] get Parkinsonian symptoms. This means that if you intervene, even at the stage where somebody has lost 30, 40, 50 percent" of the neurons.
Sapru stressed that the expressed RNAi therapy may not just be limited to familial Parkinson's disease patients, but may also prove beneficial to patients with the idiopathic form of the disease, which affects people at the later stages of life as opposed to people in their 30s and 40s as with familial Parkinson's -and is believed to be caused by a combination of genetic and environmental factors.
However, Sapru said that he and his colleagues at Northwestern are taking things one step at a time and focusing right now on familial Parkinson's disease. They are currently in talks with other academic groups about collaborating on the testing of their expressed RNAi therapy in non-human primates, and hope to be able to begin these experiments in parallel with the rodent research.
He also said that a patent application has been filed with the US Patent and Trademark Office on the RNAi therapy, and that his group is open to any possible partnerships with industry.
Doug Macron ([email protected])