NEW YORK (GenomeWeb) – Researchers from Genzyme this month reported new proof-of-concept data showing that an expressed RNAi approach could be used to both prevent and treat the manifestations of Huntington's disease (HD) in an animal model of the condition.
Though promising, the work is still early stage, cautioned Lisa Stanek, lead author of the study. And while the company is continuing to develop the RNAi-based treatment for HD, clinical studies remain years away, she said.
Huntington's disease is a member of a class of disorders known as polyglutamine diseases that is caused by the expansion of a CAG repeat in exon 1 of the gene huntingtin (Htt). The resulting extension of the polyglutamine tract in the N-terminal region confers a toxic gain-of-function to the mutant huntintin protein.
Much of the disease's pathology is believe to be associated with formation of mutant Htt-containing aggregates in the striatum of the brain, although there is evidence of cortical involvement.
Genzyme has been working in the HD field for several years, including through a collaboration with Isis Pharmaceuticals to develop an antisense-based drugs for the disease. Although the two companies were able to develop antisense oligos capable of blocking Htt expression, delivering the molecules into the brain proved a challenge as they do not readily cross the blood-brain barrier, Stanek said. Further complicating an antisense strategy to treatment is the chronic nature of HD, which requires continuous treatment for the life of the patient.
Notably, RNAi leader Alnylam Pharmaceuticals faced similar issues in its HD efforts, which centered around an alliance with Medtronic on a treatment involving the delivery of therapeutic siRNAs to the central nervous system via an infusion pump. In mid-2012, Alnylam stepped away from that arrangement, citing the program's complexity.
In order to overcome the delivery issue, Genzyme turned to AAV-enabled expressed RNAi, taking cues from University of Iowa researcher Beverly Davidson, who has long been working an on RNAi therapy for HD — including through a collaboration with gene therapy firm Targeted Genetics, which sold off its AAV assets to Genzyme in 2009.
In 2008, Davidson published a paper describing the use of microRNA scaffolds as part of an expressed RNAi approach for HD. Specifically, she and her team found that by incorporating shRNAs against Htt into an artificial miRNA backbone based on miR-30, they could achieve targeting silencing while eliminating the shRNA-related neurotoxicity that plagued previous efforts.
The Genzyme scientists began by recapitulating Davidson's findings in their own model of HD, so-called YAC128 mice. Once they had done that, they worked with the company's genomics specialists to create more robust RNAi sequences against Htt, Stanek said.
After testing the sequences in vitro, the most efficacious was embedded into an artificial miRNA backbone and cloned into a vector that would also express an enhanced GFP reporter gene, according to their latest paper, which appeared in Human Gene Therapy.
The scientists then ran two sets of experiments, one in which the RNAi agent was injected into the striata of young YAC128 mice that had not yet developed symptoms of HD and another in which the drug was delivered into the striata of older mice displaying significant disease pathology, Stanek explained.
In both cases, the Genzyme team found that the RNAi compound transduced more than 80 percent of the cells in the striatum, cutting levels of mutant and wild-type Htt by around 40 percent.
While developers of RNAi therapeutics traditionally aim for significantly higher levels of target knockdown, Stanek noted that because Genzyme's agent targets both versions of the huntingtin gene, too high a level of gene silencing can be deleterious.
"Because we don't know the long-term effects of silencing wild-type huntingtin, we actually don't want to go beyond 50 percent reduction," she said, citing data in the literature linking wild-type Htt knockdown of 80 percent or greater with toxicity. Meanwhile, non-human primate data generated by other groups suggest that mutant Htt inhibition of around 50 percent is both therapeutic and safe.
Compellingly, Stanek and her colleagues also found that the RNAi treatment prevented the onset of HD symptoms in the young mice out to five months, and that it seemingly reversed the effects of the disease in the older mice by clearing the mutant Htt aggregates that had accumulated in their striata.
"The brains of the treated animals looked essentially like wild-type animals at the end of the study," she said.
Additionally, treatment was associated with significant improvements in the behavior deficits in the older animals as determined by measuring their ability to navigate a rotating rod and perform in a swim test. Lastly, treatment was not associated with any overt neurotoxicity.
With these findings in hand, Stanek said that she and her Genzyme colleagues are now in the process of fine-tuning the therapy's RNAi payload, looking for sequences that are effective in knocking down Htt but have a low potential for off-target effects — a particular concern for a drug that would be active for years.
At the same time, they are trying to identify an AAV vector best fitted for HD — for instance, one that achieves high striatal transduction and has the potential for cortical transduction.