Huntington's disease (HD) is a fatal, neurodegenerative disorder resulting from an expanded tri-nucleotide repeat (CAG) in exon 1 of the HD gene (HTT). The glutamine expansion in the encoded protein, huntingtin (HTT) confers a toxic gain of function, causing degeneration of neurons in many brain regions, particularly in the striatum. Because these brain regions are involved with various aspects of motor programming and cognition, HD patients experience many symptoms including chorea (involuntary dance-like movements of the limbs and neck), loss of short-term memory and emotional disturbances that can include anxiety, depression and aggression. RNA interference (RNAi) has recently emerged as a leading candidate approach to reduce expression of disease genes by degrading the encoding mRNA. While normal huntingtin plays a vital role in development, we have demonstrated that a partial (60%) non-allele specific reduction in HTT expression is both well tolerated and therapeutically beneficial in adult HD transgenic mice. Here, we propose to test the efficiency, safety and therapeutic benefit of this approach in the nonhuman primate (NHP) as a pre-clinical step towards developing RNAi as a therapy for HD patients.
We first aim to characterize the efficiency and safety of non-allele specific RNAi targeting HTT mRNA in the normal NHP brain as a proof-of-principle. Rhesus macaques (n=4) will receive unilateral, stereotaxic injections into the striatum of a recombinant viral vector that expresses mi2.4 (rAAV-mi2.4), a HTT-specific microRNA. The opposite hemisphere will be injected with a control miRNA (rAAV-miMIS) that does not reduce HTT expression. Animals will undergo behavioral evaluation to assess the efficacy and tolerability of this approach. Three months post-injection, animals will be euthanized and brains will be examined for silencing efficiency and several different safety profiles. Next, we aim to establish a NHP model of HD that includes motor dysfunction, cognitive deficits and emotional manifestations. Rhesus monkeys will receive injections into the striatum of a virus expressing a fragment of mutant HTT (mHTT) with 82 CAG repeats (rAAV-mHTT82Q, n=4) or a control fragment of mHTT with only 18 repeats (rAAV-mHTT-18Q, n=4). This strategy has been previously utilized to create a successful rodent model of HD. Animals will evaluated monthly on a variety of motor, cognitive and psychological assays. Six months post- injection, animals will be euthanized and their brains will be analyzed for signs of HD pathology. Lastly, we aim to test the therapeutic benefit of RNAi in the NHP model of HD. NHPs will be co-injected into the striatum with rAAV-mi2.4 (n=4) or rAAV-miMIS (n=4) along with rAAV-mHTT82Q. Animals will be evaluated monthly on a variety of motor, cognitive and psychological assays. Upon sacrifice at 6 months post-injection, brains will be analyzed for the prevention of HD-related pathology. Together, these studies will assess the safety and therapeutic benefit of RNAi in the NHP brain. The studies proposed here, along with the didactic and applied training, will be invaluable in assisting my transition from a post-doctoral fellow to an independent investigator.
The proposed set of studies aims at testing the benefit of RNA interference as a potential therapy for Huntington's disease (HD). There is no cure for this devastating, fatal disease and currently 30,000 people are affected in the United States alone. Testing the utility of RNA interference in a nonhuman primate model of HD is a necessary and key step towards its potential use in the clinic.