Benefits of global mutant huntingtin lowering diminish over time in a Huntington's disease mouse model.

We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.

Mutant Huntingtin Is Cleared from the Brain via Active Mechanisms in Huntington Disease.

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons.SIGNIFICANCE STATEMENT: Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.

Analysis of mutant and total huntingtin expression in Huntington's disease murine models.

Huntington's disease (HD) is a monogenetic neurodegenerative disorder that is caused by the expansion of a polyglutamine region within the huntingtin (HTT) protein, but there is still an incomplete understanding of the molecular mechanisms that drive pathology. Expression of the mutant form of HTT is a key aspect of diseased tissues, and the most promising therapeutic approaches aim to lower expanded HTT levels. Consequently, the investigation of HTT expression in time and in multiple tissues, with assays that accurately quantify expanded and non-expanded HTT, are required to delineate HTT homeostasis and to best design and interpret pharmacodynamic readouts for HTT lowering therapeutics. Here we evaluate mutant polyglutamine-expanded (mHTT) and polyglutamine-independent HTT specific immunoassays for validation in human HD and control fibroblasts and use to elucidate the CSF/brain and peripheral tissue expression of HTT in preclinical HD models.

Disease-related Huntingtin seeding activities in cerebrospinal fluids of Huntington's disease patients.

In Huntington's disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon 1 reporter, which showed remarkably high sensitivity and specificity in detecting mHTT seeding species in HD patient biosamples. We showed that the seeding-competent mHTT species in HD CSF are significantly elevated upon disease onset and with the progression of neuropathological grades. Mechanistically, we showed that mHTT seeding activities in patient CSF could be ameliorated by the overexpression of chaperone DNAJB6 and by antibodies against the polyproline domain of mHTT. Together, our study developed a selective and scalable cell-based tool to investigate mHTT seeding activities in HD CSF, and demonstrated that the CSF mHTT seeding species are significantly associated with certain disease states. This seeding activity can be ameliorated by targeting specific domain or proteostatic pathway of mHTT, providing novel insights into such pathological activities.

Targeting Huntingtin Expression in Patients with Huntington's Disease.

BACKGROUND: Huntington's disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT, resulting in a mutant huntingtin protein. IONIS-HTTRx (hereafter, HTTRx) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant huntingtin. METHODS: We conducted a randomized, double-blind, multiple-ascending-dose, phase 1-2a trial involving adults with early Huntington's disease. Patients were randomly assigned in a 3:1 ratio to receive HTTRx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of huntingtin. The primary end point was safety. The secondary end point was HTTRx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant huntingtin in CSF. RESULTS: Of the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTTRx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTTRx-treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTTRx in CSF showed dose dependence up to doses of 60 mg. HTTRx treatment resulted in a dose-dependent reduction in the concentration of mutant huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and -20%, -25%, -28%, -42%, and -38% in the HTTRx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively). CONCLUSIONS: Intrathecal administration of HTTRx to patients with early Huntington's disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02519036.).

Fluid and imaging biomarkers for Huntington's disease.

Huntington's disease is a chronic progressive neurodegenerative condition for which there is no disease-modifying treatment. The known genetic cause of Huntington's disease makes it possible to identify individuals destined to develop the disease and instigate treatments before the onset of symptoms. Multiple trials are already underway that target the cause of HD, yet clinical measures are often insensitive to change over typical clinical trial duration. Robust biomarkers of drug target engagement, disease severity and progression are required to evaluate the efficacy of treatments and concerted efforts are underway to achieve this. Biofluid biomarkers have potential advantages of direct quantification of biological processes at the molecular level, whilst imaging biomarkers can quantify related changes at a structural level in the brain. The most robust biofluid and imaging biomarkers can offer complementary information, providing a more comprehensive evaluation of disease stage and progression to inform clinical trial design and endpoints.

Evaluation of mutant huntingtin and neurofilament proteins as potential markers in Huntington's disease.

Huntington's disease (HD) is a genetic progressive neurodegenerative disorder, caused by a mutation in the HTT gene, for which there is currently no cure. The identification of sensitive indicators of disease progression and therapeutic outcome could help the development of effective strategies for treating HD. We assessed mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations in cerebrospinal fluid (CSF) and blood in parallel with clinical evaluation and magnetic resonance imaging in premanifest and manifest HD mutation carriers. Among HD mutation carriers, NfL concentrations in plasma and CSF correlated with all nonbiofluid measures more closely than did CSF mHTT concentration. Longitudinal analysis over 4 to 8 weeks showed that CSF mHTT, CSF NfL, and plasma NfL concentrations were highly stable within individuals. In our cohort, concentration of CSF mHTT accurately distinguished between controls and HD mutation carriers, whereas NfL concentration, in both CSF and plasma, was able to segregate premanifest from manifest HD. In silico modeling indicated that mHTT and NfL concentrations in biofluids might be among the earliest detectable alterations in HD, and sample size prediction suggested that low participant numbers would be needed to incorporate these measures into clinical trials. These findings provide evidence that biofluid concentrations of mHTT and NfL have potential for early and sensitive detection of alterations in HD and could be integrated into both clinical trials and the clinic.

Dose-Dependent Lowering of Mutant Huntingtin Using Antisense Oligonucleotides in Huntington Disease Patients.

On December 11 of 2017, Ionis Pharmaceuticals published a press release announcing dose-dependent reductions of mutant huntingtin protein in their HTTRx Phase 1/2a study in Huntington disease (HD) patients. The results from this Ionis trial have gained much attention from the patient community and the oligonucleotide therapeutics field, since it is the first trial targeting the cause of HD, namely the mutant huntingtin protein, using antisense oligonucleotides (ASOs). The press release also states that the primary endpoints of the study (safety and tolerability) were met, but does not contain data. This news follows the approval of another therapeutic ASO nusinersen (trade name Spinraza) for a neurological disease, spinal muscular atrophy, by the U.S. Food and Drug Administration and European Medicines Agency, in 2016 and 2017, respectively. Combined, this offers hope for the development of the HTTRx therapy for HD patients.

A Critical Evaluation of Wet Biomarkers for Huntington's Disease: Current Status and Ways Forward.

There is an unmet clinical need for objective biomarkers to monitor disease progression and treatment response in Huntington's disease (HD). The aim of this review is, therefore, to provide practical advice for biomarker discovery and to summarise studies on biofluid markers for HD. A PubMed search was performed to review literature with regard to candidate saliva, urine, blood and cerebrospinal fluid biomarkers for HD. Information has been organised into tables to allow a pragmatic approach to the discussion of the evidence and generation of practical recommendations for future studies. Many of the markers published converge on metabolic and inflammatory pathways, although changes in other analytes representing antioxidant and growth factor pathways have also been found. The most promising markers reflect neuronal and glial degeneration, particularly neurofilament light chain. International collaboration to standardise assays and study protocols, as well as to recruit sufficiently large cohorts, will facilitate future biomarker discovery and development.

Validation of Ultrasensitive Mutant Huntingtin Detection in Human Cerebrospinal Fluid by Single Molecule Counting Immunoassay.

BACKGROUND: The measurement of disease-relevant biomarkers has become a major component of clinical trial design, but in the absence of rigorous clinical and analytical validation of detection methodology, interpretation of results may be misleading. In Huntington's disease (HD), measurement of the concentration of mutant huntingtin protein (mHTT) in cerebrospinal fluid (CSF) of patients may serve as both a disease progression biomarker and a pharmacodynamic readout for HTT-lowering therapeutic approaches. We recently published the quantification of mHTT levels in HD patient CSF by a novel ultrasensitive immunoassay-based technology and here analytically validate it for use. OBJECTIVE: This work aims to analytically and clinically validate our ultrasensitive assay for mHTT measurement in human HD CSF, for application as a pharmacodynamic biomarker of CNS mHTT lowering in clinical trials. METHODS: The single molecule counting (SMC) assay is an ultrasensitive bead-based immunoassay where upon specific recognition, dye-labeled antibodies are excited by a confocal laser and emit fluorescent light as a readout. The detection of mHTT by this technology was clinically validated following established Food and Drug Administration and European Medicine Agency guidelines. RESULTS: The SMC assay was demonstrated to be accurate, precise, specific, and reproducible. While no matrix influence was detected, a list of interfering substances was compiled as a guideline for proper collection and storage of patient CSF samples. In addition, a set of recommendations on result interpretation is provided. CONCLUSIONS: This SMC assay is a robust and ultrasensitive method for the relative quantification of mHTT in human CSF.

Huntington Disease: Linking Pathogenesis to the Development of Experimental Therapeutics.

Huntington disease (HD) is an autosomal dominant neurodegenerative condition caused by a CAG trinucleotide expansion in the huntingtin gene. At present, the HD field is experiencing exciting times with the assessment for the first time in human subjects of interventions aimed at core disease mechanisms. Out of a portfolio of interventions that claim a potential disease-modifying effect in HD, the target huntingtin has more robust validation. In this review, we discuss the spectrum of huntingtin-lowering therapies that are currently being considered. We provide a critical appraisal of the validation of huntingtin as a drug target, describing the advantages, challenges, and limitations of the proposed therapeutic interventions. The development of these new therapies relies strongly on the knowledge of HD pathogenesis and the ability to translate this knowledge into validated pharmacodynamic biomarkers. Altogether, the goal is to support a rational drug development that is ethical and cost-effective. Among the pharmacodynamic biomarkers under development, the quantification of mutant huntingtin in the cerebral spinal fluid and PET imaging targeting huntingtin or phosphodiesterase 10A deserve special attention. Huntingtin-lowering therapeutics are eagerly awaited as the first interventions that may be able to change the course of HD in a meaningful way.