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Lowering mutant Huntingtin is a consensus therapeutic strategy for Huntington’s disease. In this issue of Neuron, Kordasiewicz et al. (2012) show the benefit of transient antisense oligonucleotide (ASO) therapy to degrade Huntingtin mRNA and elicit sustained therapeutic benefit in HD mice.
Huntington’s disease (HD) is one of the most common dominantly inherited neurodegenerative disorders, characterized by a clinical triad of movement disorder, cognitive deficits, and psychiatric symptoms. The average age of onset for HD is around 40 years old. HD is relentlessly progressive and patients eventually succumb to disease complications about 20 years after symptom onset. HD neuropathology is characterized by selective and massive degeneration of the striatal neurons and to a lesser extent the deep-layer cortical neurons; however, other brain regions such as the thalamus, hippocampus, and white matter (e.g., corpus callosum) are also affected in patients. Currently, there is no effective therapy to prevent the onset or slow the progression of HD.
Because of its monogenetic etiology, HD is a tractable model to study pathogenesis and develop rational therapeutics for a neurodegenerative disorder. HD is caused by a CAG repeat expansion encoding an elongated polyglutamine (polyQ) repeat near the N terminus of the Huntingtin (Htt) protein. The precise molecular functions of Htt remain incompletely understood, but it is essential for embryonic development and adult neuronal survival, at least in mice (e.g., Dragatsis et al., 2000). Studies in a plethora of model systems have yielded numerous potential pathogenic pathways and targets that could modify mutant Htt (mHtt)-induced phenotypes (Ross and Tabrizi, 2011). Several such pathways appear to exert large disease-suppressing effects in animal models (Ross and Tabrizi, 2011), but candidate therapies targeting these pathways remain to be developed.
Although consensus molecular targets that can counteract the toxic consequences of mHtt are yet to emerge, an unequivocal target for HD therapy is mHtt itself. HD presents a prime opportunity to test the hypothesis that lowering levels of a toxic disease-causing protein in proper cell types and disease stages should have a large therapeutic effect. The proof-of-concept experiment to support such a notion came from a conditional, tet-regulatable mouse model expressing mHtt exon1 fragment, in which shutting down mHtt fragment expression after disease onset leads to a reversal of behavioral deficits, neurodegenerative pathology, and mHtt aggregation (Yamamoto et al., 2000). However, lowering Htt as a therapeutic strategy is not without potential risks. In mice, conditional deletion of endogenous Htt in the forebrain neurons results in progressive neurodegeneration (Dragatsis et al., 2000), suggesting that a minimal level of Htt may be necessary for the survival of certain adult neurons.
While theoretically mHtt can be targeted at the levels of DNA, RNA, or protein, the most advanced Htt-lowering therapeutics to date have been directed toward Htt messenger RNA (mRNA). The first successful strategy to reduce Htt mRNA was through RNA interference (RNAi) by the Davidson group (Harper et al., 2005), in which striatal injections ofadeno-associated virus (AAV) expressing a short hairpin RNA (shRNA) lead to a reduction of mHtt and its aggregates and amelioration of motor deficits in an mHtt fragment model. Subsequent improvements of the strategy resulted in AAV-mediated delivery of a less toxic but equally efficacious artificial microRNA (miRNA) against mHtt (McBride et al., 2008). A second RNAi strategy, by direct striatal infusion of synthetic small interfering RNA (siRNA) duplex, has also shown efficacy in mitigating mHtt fragment toxicities in striatal neurons (DiFiglia et al., 2007). These RNAi strategies lay a solid foundation for Htt-lowering therapies for HD. However, several lingering questions remain to be addressed. First, can such a therapy maintain its efficacy and safety profiles in situations requiring chronic administration, such as in the more slowly progressive full-length mHtt mouse models? Second, are there alternative ways to deliver Htt-lowering therapy to broader brain regions and cell types beyond the striatum that may also contribute to symptoms of HD?
A study in this issue of Neuron by Kordasiewicz et al. (2012) provides strong preclinical evidence to support the use of antisense oligonucleotides (ASOs) as an Htt-lowering therapeutic for HD. ASOs are single-stranded DNA oligonucleotides (usually 8–50 nucleotides) that target cellular mRNA transcripts via complementary base pairing. The resulting DNA/RNA duplex undergoes catalytic degradation of the RNA component by RNase H, an enzyme present in most mammalian cells. Importantly, the single-stranded ASO can be recycled to mediate multiple rounds of selective mRNA degradation (Figure 1A). The stability and potency of ASOs are due to the phosphorothioate backbone and 2′-O-methoxyethyl (MOE) deoxynucleotide (DNA) sugar modifications, with specificity conferred by bioinformatic analysis and cell-based screening to optimize target engagement while minimizing off-target toxicity (Bennett and Swayze, 2010). A strength for ASOs as candidate therapeutic agents is the safety profiles in human studies so far, with one approved drug in clinical use and another 35 in clinical development (Bennett and Swayze, 2010). Indeed, one such clinical study is a phase I clinical trial of ASO-mediated lowering of mutant SOD1 in familial amyotrophic lateral sclerosis, based on the original preclinical study by Cleveland and colleagues (Smith et al., 2006).
To test ASO therapy in HD models, Kordasiewicz et al. (2012) first established drug-like properties for the Htt ASOs. In the BACHD model that expresses full-length human mHtt (Gray et al., 2008), a 2 week infusion of two separate ASOs into the right ventricle, one selectively targeting human and the other targeting both human and murine Htt, is sufficient to induce dose-dependent and selective reduction of Htt for up to 12 weeks, with Htt levels returning to baseline at 16 weeks. The stability and high potency of chemically modified ASOs probably contribute to the lengthy period of Htt lowering after transient ASO infusion. The second surprising finding from the pharmacokinetics study is the broad distribution of ASOs in many brain regions (e.g., cortex, striatum, thalamus, midbrain, and brainstem) from intraventricular ASO delivery. Such broad diminution of mHtt synthesis in multiple brain regions may be advantageous in treating HD, since the ubiquitously expressed mHtt is likely to affect multiple brain regions to cause the core clinical symptoms of HD.
A strong point of the current study is the use of three distinct transgenic mouse models of HD, three Htt-targeting ASOs, and seven independent preclinical trials to demonstrate the efficacy of ASOs in abating disease phenotypes in vivo. In R6/2, an mHtt-exon1 transgenic mouse model that exhibits aggressive and lethal disease course, 4 week infusion of ASOs at a symptomatic stage leads to 60% lowering of mHtt exon1, amelioration of brain atrophy, and prolonged survival. However, the nuclear inclusion formation was not modified by ASO treatment, suggesting only partial improvement of disease pathology in this model.
The therapeutic efficacy of Htt ASOs was more thoroughly investigated in two full-length human mHtt genomic transgenic mouse models, YAC128 and BACHD. In the YAC128 model, which expresses human full-length mHtt with 128Q (Slow et al., 2003), 2 week ASO infusion results in 80% mHtt lowering and significant improvement of motor coordination on rotarod test. However, treatment initiated at a later and more symptomatic age (6 months) leads only to a trend, but not statistically significant improvement, suggesting that earlier ASO treatment may confer better therapeutic effect, at least in this model.
The most in-depth preclinical assessments the authors performed with ASOs were conducted in BACHD mice, which express full-length human mHtt with 97Q under endogenous genomic regulation (Gray et al., 2008). BACHD mice exhibit progressive motor and psychiatric-like behavioral deficits (e.g., anxiety), selective cortical and striatal atrophy, and confer good statistical power to detect disease modification (Gray et al., 2008). With 2 week intraventricular infusion of human-selective ASOs in BACHD mice at 6 months of age, the treated mice show significant improvement in motor coordination and open-field exploration and reduction in anxiety at 8–12 months of age. To further evaluate the potential lasting beneficial effects of transient ASO therapy, Kordasiewicz et al. (2012) performed a second BACHD trial to infuse ASOs at 6 months and assayed these mice up to 15 months of age. Surprisingly, even 9 months after ASO infusion and 5 months after mHtt level returns to baseline, ASO-treated BACHD mice still show sustained benefits in motor and anxiety behaviors. The results from neuropathology are somewhat mixed, with ASO-treated BACHD mice showing fewer mHtt aggregates, but no rescue of the brain atrophy phenotype. The latter finding raises some questions about whether earlier ASO delivery or a repeated treatment regimen may be necessary to ameliorate neurodegeneration.
In this tour de force preclinical study, Kordasiewicz et al. (2012) also addressed the issue of the safety and efficacy of targeting endogenous Htt. They showed that the infusion of ASOs against both human and murine Htt into BACHD mice, achieving up to a 75% reduction of human mHtt and murine wild-type Htt, did not alter therapeutic benefits. Moreover, in wild-type mice, reducing Htt by about 75% did not elicit any aberrant motor phenotypes. A final crucial piece of preclinical information was the intrathecal infusion of ASOs into rhesus monkeys, showing that Htt could be reduced in some of the brain regions affected in HD (e.g., cortex) but not others (e.g., caudate). Intrathecal infusion, a much less invasive method compared to intraventricular or intraparenchymal injection, is already approved for ASO delivery in the ongoing ALS trial. While the current study did not provide any safety data on the infusion of Htt ASOs in the nonhuman primates, necessary for further clinical development, the work of Kordasiewicz et al. (2012) presents an important preclinical demonstration of the reproducible benefit of ASO-mediated Htt-lowering therapy in multiple HD mouse models.
The most surprising and important finding from the current study is the sustained benefit of transient mHtt lowering, with multiple phenotypic improvements well beyond the period of disease target suppression. This phenomenon has been referred to as a “Huntingtin Holiday” by Carl Johnson, the Scientific Director of the Hereditary Disease Foundation (Figure 1B). The precise mechanisms underlying this remarkable effect remain unknown and should be investigated. This finding does suggest that in HD mice, and likely in patients, critical disease symptoms may arise from reversible neuronal dysfunction, and transient relief of the primary insult may help the affected neurons to better handle the re-expressed mHtt. The Huntingtin Holiday effect also points to a potential clinical trial design with periodic infusion of Htt-lowering therapy.
With Htt-lowering therapies primed for clinical studies in HD, several pressing issues remain to be clarified. First and foremost, we need to know when in the disease course and where in the brain such therapies should be delivered. The current study supports the intuition that early ASO delivery may confer more benefit to modify the disease course. The question of where in the brain Htt-lowering therapy should be delivered is not yet resolved, but current models support that mHtt in multiple cell types may contribute to the disease (Gu et al., 2005). Delineating precise cell-type contributions to HD phenotypes will be crucial to select optimal Htt-lowering agents and delivery strategies for clinical trials. The second question is whether both mutant and wild-type Htt alleles should be targeted indiscriminately, or if allele-specific silencing is a better choice. The latter strategy may minimize potential toxicity due to lowering of endogenous Htt in human, which may not be predicted from animal studies. To this end, the welcome news is that only a few single-nucleotide polymorphisms may be able to distinguish the majority of HD patient alleles from control alleles, and allelic-specific silencing can be achieved with siRNAs or ASOs (e.g., Pfister et al., 2009; Carroll et al., 2011). The third question is the urgent need to develop biomarkers that can report on the central lowering of mHtt and early reversal of neuronal dysfunction. Finally, the most difficult question, and one still worth pondering, is how well successful preclinical studies of Htt-lowering therapies will translate into HD clinical trials. Species differences aside, one should keep in mind that HD mouse models only recapitulate a subset of the complex clinical phenotypes of the patients, and most Htt-lowering therapies so far have shown partial but not full disease reversal in such models. Keeping such limitations in mind, the consistent benefit of Htt-lowering therapy across different therapeutic reagents and model platforms, as evidenced from the current study, will undoubtedly energize the field to further pursue such innovative and rational therapies for HD.