Delivery of mutant huntingtin-lowering antisense oligonucleotides to the brain by intranasally administered apolipoprotein A-I nanodisks.

Lowering mutant huntingtin (mHTT) in the central nervous system (CNS) using antisense oligonucleotides (ASOs) is a promising approach currently being evaluated in clinical trials for Huntington disease (HD). However, the therapeutic potential of ASOs in HD patients is limited by their inability to cross the blood-brain barrier (BBB). In non-human primates, intrathecal infusion of ASOs results in limited brain distribution, with higher ASO concentrations in superficial regions and lower concentrations in deeper regions, such as the basal ganglia. To address the need for improved delivery of ASOs to the brain, we are evaluating the therapeutic potential of apolipoprotein A-I nanodisks (apoA-I NDs) as novel delivery vehicles for mHTT-lowering ASOs to the CNS after intranasal administration. Here, we have demonstrated the ability of apoA-I nanodisks to bypass the BBB after intranasal delivery in the BACHD model of HD. Following intranasal administration of apoA-I NDs, apoA-I protein levels were elevated along the rostral-caudal brain axis, with highest levels in the most rostral brain regions including the olfactory bulb and frontal cortex. Double-label immunohistochemistry indicates that both the apoA-I and ASO deposit in neurons. Most importantly, a single intranasal dose of apoA-I ASO-NDs significantly reduces mHTT levels in the brain regions most affected in HD, namely the cortex and striatum. This approach represents a novel non-invasive means for improving delivery and brain distribution of oligonucleotide therapies and enhancing likelihood of efficacy. Improved ASO delivery to the brain has widespread application for treatment of many other CNS disorders.

Cerebrospinal fluid mutant huntingtin is a biomarker for huntingtin lowering in the striatum of Huntington disease mice.

Huntington disease (HD) is a neurodegenerative disease caused by a trinucleotide repeat expansion in the HTT gene encoding an elongated polyglutamine tract in the huntingtin (HTT) protein. Expanded mutant HTT (mHTT) is toxic and leads to regional atrophy and neuronal cell loss in the brain, which occurs earliest in the striatum. Therapeutic lowering of mHTT in the central nervous system (CNS) has shown promise in preclinical studies, with multiple approaches currently in clinical development for HD. Quantitation of mHTT in the cerebrospinal fluid (CSF) is being used as a clinical pharmacodynamic biomarker of target engagement in the CNS. We have previously shown that the CNS is a major source of mHTT in the CSF. However, little is known about the specific brain regions and cell types that contribute to CSF mHTT. Therefore, a better understanding of the origins of CSF mHTT and whether therapies targeting mHTT in the striatum would be expected to be associated with significant lowering of mHTT in the CSF is needed. Here, we use complementary pharmacological and genetic-based approaches to either restrict expression of mHTT to the striatum or selectively deplete mHTT in the striatum to evaluate the contribution of this brain region to mHTT in the CSF. We show that viral expression of a mHTT fragment restricted to the striatum leads to detectable mHTT in the CSF. We demonstrate that targeted lowering of mHTT selectively in the striatum using an antisense oligonucleotide leads to a significant reduction of mHTT in the CSF of HD mice. Furthermore, using a transgenic mouse model of HD that expresses full length human mHTT and wild type HTT, we show that genetic inactivation of mHTT selectively in the striatum results in a significant reduction of mHTT in the CSF. Taken together, our data supports the conclusion that the striatum contributes sufficiently to the pool of mHTT in the CSF that therapeutic levels of mHTT lowering in the striatum can be detected by this measure in HD mice. This suggests that CSF mHTT may represent a pharmacodynamic biomarker for therapies that lower mHTT in the striatum.

Reliable Resolution of Full-Length Huntingtin Alleles by Quantitative Immunoblotting.

BACKGROUND: Therapeutics that lower mutant huntingtin (mHTT) have shown promise in preclinical studies and are in clinical development for the treatment of Huntington disease (HD). Multiple assays have been developed that either quantify mHTT or total HTT but may not accurately measure levels of wild type HTT (wtHTT) in biological samples. OBJECTIVE: To optimize a method that can be used to resolve, quantify and directly compare levels of full length wtHTT and mHTT in HD samples. METHODS: We provide a detailed quantitative immunoblotting protocol to reproducibly resolve full length wtHTT and mHTT in multiple HD mouse and patient samples. RESULTS: We show that this assay can be modified, depending on the sample, to resolve wtHTT and mHTT with a wide range of polyglutamine length differences (ΔQs 22-179). We also demonstrate that this method can be used to quantify allele-selective lowering of mHTT using an antisense oligonucleotide in HD patient-derived cells. CONCLUSION: This quantitative immunoblotting method can be used to reliably resolve full-length HTT alleles with ΔQs≥22 and allows for direct comparison of wtHTT and mHTT levels in HD samples.

Interrupting sequence variants and age of onset in Huntington's disease: clinical implications and emerging therapies.

BACKGROUND: Huntington's disease is a fatal neurodegenerative disorder that is caused by CAG-CAA repeat expansion, encoding polyglutamine, in the huntingtin (HTT) gene. Current age-of-clinical-onset prediction models for Huntington's disease are based on polyglutamine length and explain only a proportion of the variability in age of onset observed between patients. These length-based assays do not interrogate the underlying genetic variation, because known genetic variants in this region do not alter the protein coding sequence. Given that individuals with identical repeat lengths can present with Huntington's disease decades apart, the search for genetic modifiers of clinical age of onset has become an active area of research. RECENT DEVELOPMENTS: Results from three independent genetic studies of Huntington's disease have shown that glutamine-encoding CAA variants that interrupt DNA CAG repeat tracts, but do not alter polyglutamine length or polyglutamine homogeneity, are associated with substantial differences in age of onset of Huntington's disease in carriers. A variant that results in the loss of CAA interruption is associated with early onset and is particularly relevant to individuals that carry alleles in the reduced penetrance range (ie, CAG 36-39). Approximately a third of clinically manifesting carriers of reduced penetrance alleles, defined by current diagnostics, carry this variant. Somatic repeat instability, modified by interrupted CAG tracts, is the most probable cause mediating this effect. This relationship is supported by genome-wide screens for disease modifiers, which have revealed the importance of DNA-repair genes in Huntington's disease (ie, FAN1, LIG1, MLH1, MSH3, PMS1, and PMS2). WHERE NEXT?: Focus needs to be placed on refining our understanding of the effect of the loss-of-interruption and duplication-of-interruption variants and other interrupting sequence variants on age of onset, and assessing their effect in disease-relevant brain tissues, as well as in diverse population groups, such as individuals from Africa and Asia. Diagnostic tests should be augmented or updated, since current tests do not assess the underlying DNA sequence variation, especially when assessing individuals that carry alleles in the reduced penetrance range. Future studies should explore somatic repeat instability and DNA repair as new therapeutic targets to modify age of onset in Huntington's disease and in other repeat-mediated disorders. Disease-modifying therapies could potentially be developed by therapeutically targeting these processes. Promising approaches include therapeutically targeting the expanded repeat or directly perturbing key DNA-repair genes (eg, with antisense oligonucleotides or small molecules). Targeting the CAG repeat directly with naphthyridine-azaquinolone, a compound that induces contractions, and altering the expression of MSH3, represent two viable therapeutic strategies. However, as a first step, the capability of such novel therapeutic approaches to delay clinical onset in animal models should be assessed.

Potent and sustained huntingtin lowering via AAV5 encoding miRNA preserves striatal volume and cognitive function in a humanized mouse model of Huntington disease.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a pathogenic expansion of a CAG repeat in the huntingtin (HTT) gene. There are no disease-modifying therapies for HD. Artificial microRNAs targeting HTT transcripts for degradation have shown preclinical promise and will soon enter human clinical trials. Here, we examine the tolerability and efficacy of non-selective HTT lowering with an AAV5 encoded miRNA targeting human HTT (AAV5-miHTT) in the humanized Hu128/21 mouse model of HD. We show that intrastriatal administration of AAV5-miHTT results in potent and sustained HTT suppression for at least 7 months post-injection. Importantly, non-selective suppression of huntingtin was generally tolerated, however high dose AAV5-miHTT did induce astrogliosis. We observed an improvement of select behavioural and modest neuropathological HD-like phenotypes in Hu128/21 mice, suggesting a potential therapeutic benefit of miRNA-mediated non-selective HTT lowering. Finally, we also observed that potent reduction of wild type HTT (wtHTT) in Hu21 control mice was tolerated up to 7 months post-injection but may induce impairment of motor coordination and striatal atrophy. Taken together, our data suggests that in the context of HD, the therapeutic benefits of mHTT reduction may outweigh the potentially detrimental effects of wtHTT loss following non-selective HTT lowering.