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Accelerating Therapeutic Development for Huntington's Disease

2022 Conference

CHDI’s 17th Annual HD Therapeutics Conference was back in-person after a year-long hiatus, and took place February 28 – March 3, 2022. This unique conference series focuses on drug discovery and development for Huntington’s disease, and draws participants from the biotech and pharmaceutical sectors as well as academia. The conference is intended as a forum where all participants can share ideas, learn about new disciplines, network with colleagues and build new collaborative partnerships. We are indebted to all of the conference speakers, and especially grateful to those who are able to make their presentations available here for a wider audience.

Simple Complex repeats and disease severity in Huntington disease


Darren G Monckton, PhD, University of Glasgow

Huntington disease (HD) is caused by the expansion of an apparently simple polyglutamine-encoding CAG repeat in the HTT gene. The more CAG repeats a person inherits the earlier the disease onset – simple. However, it has become apparent that the HTT polyglutamine encoding repeat is not actually so simple at the sequence level. In disease-associated alleles the CAG repeat is usually proceeded by an additional polyglutamine-encoding CAA CAG cassette. However, in HD populations of European ancestry there are some rare disease-associated alleles in which this polyglutamine-encoding CAA CAG cassette has either been duplicated or lost. These rare individuals (~ 3%) have been used to establish that the critical factor driving disease onset is the number of pure CAG repeats, not the total number of polyglutamine-encoding CAG and CAA repeats. Notably, the rate of somatic expansion is also best predicted by the number of pure CAG repeats, consistent with a model in which somatic expansion is a key second step driving disease onset. Nonetheless, although correcting for pure CAG length dramatically improves the genotype-phenotype associations, individuals inheriting disease-associated alleles with loss of the CAA CAG cassette still appear to have disease onset ~10 years earlier than expected. Recently, we have sequenced the HTT repeat in a South African HD cohort with black African ancestry. The majority of black South African HD cases also carry the the polyglutamine-encoding CAA CAG cassette. However, ~45% of black South African HD cases carry an allele in which a proline-encoding CCA codon, two codons down from the polyglutamine encoding CAG/ CAA repeat, is absent. Very surprisingly, despite having no effect on the polyglutamine or proline-encoding potential of the allele, this structure was associated with a ~4-years earlier age at diagnosis of HD in South African populations. By sequencing ~5,000 HD individuals of European ancestry from the Enroll-HD cohort this effect appears to be confirmed. Moreover, we show that the apparent disease accelerating residual effect of loss of the CAA CAG cassette appears to be driven by loss of the downstream CCA proline-codon. Clearly, the HTT repeat is even more complex than we previously imagined.

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Single-cell-resolution analysis of Huntington’s disease pathogenesis and CAG-repeat expansion in human brain

Steve McCarroll, PhD, Harvard Medical School & Broad Institute

A critical need in Huntington’s disease (HD) research is to understand the pathophysiological process – the series of molecular and cellular events by which inherited CAG-repeat expansions in the huntingtin (HTT) gene lead to disease. To address the need to identify such mechanisms in HD and other brain illnesses, we developed a technology—droplet-based single-cell RNA-seq—to profile the molecular states of thousands of individual brain cells by measuring RNA expression at single-cell resolution and in all cell types at once. To better understand HD pathogenesis, we have applied this approach to analyze RNA expression in more than one million cell nuclei from the caudate and cerebral cortex of some 100 brain donors, including HD gene-expansion carriers (at a wide variety of disease stages) and controls. The resulting data help reveal distinct pathological trajectories in each cell type; distinguish universal from common from occasional cellular changes in HD; and map the effects of certain genetic modifiers to specific cell types.
CAG repeats in HTT exhibit substantial somatic expansion and length mosaicism. Human genetic studies of age-of-onset implicate multiple DNA damage response genes (HD-GeM Consortium, 2020), suggesting that somatic expansion of the CAG repeat may be an onset-determining factor. To study the cell-type specificity and potential biological consequences of CAG-repeat expansion, we developed a laboratory and companion computational approach to measure the length of the HTT CAG-repeat expansion in thousands of individual cells alongside these cells’ larger, genome-wide patterns of RNA expression. We can now identify and characterize individual cells in which CAG repeats have expanded and to what degree. Early results from applying this approach reveal profound cell-type specificity in somatic CAG expansions, providing biological evidence for a model that connects somatic CAG expansion to cellular vulnerability.

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Connecting uninterrupted CAG repeat in human mutant huntingtin to striatum-selective pathogenesis in HD mice

X William Yang, MD, PhD, University of California, Los Angeles

In Huntington’s disease (HD), the uninterrupted CAG-repeat length, but not the polyglutamine length, predicts disease onset. However, the underlying pathobiology remains unclear. Here, we developed a human mutant huntingtin (mHTT) BAC transgenic mouse model with long uninterrupted, and somatically unstable, CAG repeats (i.e. BAC-CAG). We performed longitudinal behavioral, neuropathological, and molecular phenotyping of the BAC-CAG model and compared its phenotypes with those observed in previous human mHTT genomic transgenic models with CAA-interruptions in the polyglutamine coding region. Our study reveals a critical role of the uninterrupted CAG repeats, beyond its encoded polyglutamine protein, in eliciting striatum-selective pathogenesis in vivo. By analyzing the distinct pathogenic entities emanated from mHTT genomic transgenes and enriched in the uninterrupted CAG-repeat model, we identified that somatic CAG-repeat instability and nuclear mHTT aggregation are best correlated with striatum-selective transcriptionopathy and behavioral impairment, while RNA-associated pathologies and repeat-associated non-AUG (RAN) translation may play less selective or late pathogenic roles, respectively. Importantly, our study demonstrates CAG-repeat purity is a key determinant of phenotypic overlap between human genomic transgenic and murine knockin models of HD. Moreover, BAC-CAG offers a unique platform to test the therapeutic synergy of targeting human mHTT (or patient-associated SNPs) and other molecular targets related to CAG-repeat instability.

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The incomplete splicing of huntingtin: implications for therapeutic approaches

Gillian Bates, PhD, Queen Square Institute of Neurology, University College London

The huntingtin gene contains 67 exons and in the presence of an expanded CAG repeat, exon 1 does not always splice to exon 2 resulting in the polyadenylated transcript, HTT1a. This mRNA terminates at cryptic polyA sites located at 680 and 1145 bp into intron 1 of mouse Htt and 2710 and 7327 bp into intron 1 of human HTT. It is present in all knock-in HD mouse models, YAC128 mice and HD post-mortem brains. HTT1a encodes the aggregation-prone and highly pathogenic exon 1 HTT protein. Given that the extent of incomplete splicing increases with increasing CAG repeat length, the HTT1a transcript and exon 1 HTT protein may be the effectors through which somatic CAG-repeat expansion exerts its pathogenic consequences.
We have developed HTT bioassays to detect soluble and aggregated HTT protein isoforms and demonstrate that the level of exon 1 HTT, and not full-length HTT, decreases with increasing HTT aggregation. We have genetically modified HdhQ150 mice to unravel the pathogenic contributions of full-length mutant HTT and exon 1 HTT. A 20 kb intron 1 deletion removed all cryptic polyA sites preventing transcription from terminating in intron 1 and the generation of HTT1a. This intronic deletion had no effect on Htt expression in wild type mice. Exon 1 HTT protein levels were almost ablated from HdhQ150 delta intron mice (HdhQ150ΔI) whilst full-length HTT levels were unchanged. The deposition of HTT aggregates in HdhQ150ΔI brains was considerably delayed over a 17-month period.
In YAC128 brains, full-length HTT and HTT1a mRNAs were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. The formation of these clusters is most likely driven by human intron 1 HTT sequences. They may play a pathogenic role, or alternatively, may be protective by preventing HTT1a from being translated in the cytoplasm to produce exon 1 HTT. Taken together, these data have implications for HTT-lowering strategies; it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering approaches on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.

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The vicious cycle linking CAG expansions to proteostasis collapse in Huntington’s disease

Presentation not posted at presenter’s request

Judith Frydman, PhD, Stanford University

How CAG-repeat expansions in huntingtin (HTT) cause Huntington’s disease (HD) remains unclear. Although Htt protein is over 3,000 amino acids long, toxicity appears linked to exon 1. Mutant Htt-ex1 (mHtt-ex1) protein fragments are detected in affected patient tissues, and ectopic expression of mHtt-ex1 in cultured cells and animal models induces HD-like toxicity. mHtt-ex1 fragments can arise from aberrant splicing yielding an mRNA variant translated into mHtt-ex1 protein as well as post-translational caspase cleavage.
Exactly how CAG expansions in a single gene can cause widespread impairment of multiple cellular processes, including DNA repair, transcription, splicing, translation, nuclear import, vesicular trafficking, mitochondrial function and stress responses, remains an outstanding and important question. HD ultimately disrupts proteostasis, which is key to maintaining a healthy functional cell.
We find CAG expansions cause an elongation rate conflict during HTT translation, when ribosomes rapidly decoding the optimal CAG/polyglutamine (polyQ) tract encounter a slowly-decoded non-optimal flanking polyproline tract. The ensuing ribosome collisions activate ribosome quality control (RQC) pathways, including ribosome ubiquitination and eIF2a phosphorylation. Because Htt synthesis is controlled by an upstream open reading frame (uORF), RQC- and stress-induced eIF2a phosphorylation increases Htt translation thus enhancing ribosome collision risk. Mutant Htt further exacerbates ribotoxic stress by depleting elongation factor eIF5A from polysomes. eIF5A loss leads to impaired cotranslational proteostasis, disrupted polyamine metabolism and a maladaptive stress response. This escalating cascade of stress and dysfunction culminates in the proteostasis collapse observed in HD. The activation of ribotoxic pathways ultimately resulting in a vicious circle of dysfunction leading to proteostasis collapse.

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HD mechanisms during brain development

Sandrine Humbert, PhD, INSERM, Grenoble Institute Neurosciences

Huntington disease (HD) is a dominantly inherited neurological disorder characterized by the dysfunction and death of neurons from the cortex and the striatum. Striatal degeneration in HD is due, at least in part, to defective cortical signaling to the striatum. Symptoms in HD typically do not appear until mid-life or later, although there is a rare juvenile-onset form of the disease. Yet huntingtin, the protein mutated in HD, and mutant huntingtin are expressed from the very beginning of life and huntingtin is essential for mouse development. Anyway, given the adult onset and dysfunction and death of adult neurons characterizing HD, most studies have focused on the toxic effects elicited by mutant huntingtin in adult post-mitotic neurons and the roles of the wild-type protein during development have been less studied.
I will discuss how the huntingtin protein regulates several steps of mouse cortical development. Huntingtin maintains the pool of cycling progenitors, ensures the multipolar-bipolar transition of newborn neurons, their proper migration and maturation. In HD mice, mutant huntingtin causes mitotic spindle misorientation of dividing progenitors and decreases cortical thickness. Mutant huntingtin also interferes with the migration and maturation of post-mitotic neurons. I will also show that, as in HD mouse models, mutant huntingtin reduces the number of proliferating cells and triggers more neural progenitors to enter lineage specification prematurely in human HD mutation carrier fetuses. Finally, I will discuss how early axonal growth defects in the developing cortex contribute to presymptomatic HD signs and consider the viewing of HD with a neurodevelopmental component.

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A structure-function analysis of HTT exon1 in a knock-in stem cell platform

Raffaele Iennaco, PhD, University of Milan & Istituto Nazionale di Genetica Molecolare

Our HTT structure-function study is built on the power of a novel stem cell platform to address a wide range of questions related to the impact of different huntingtin (HTT) protein variants—carrying combinations/deletions of exon1 domains, CAG repeats, and/or critical amino acids for post-translational HTT modifications—on neuronal cell biology. To this aim, we engineered a mouse embryonic stem cell (mESC) line to insert a recombination-mediated cassette exchange (RMCE) flanking the endogenous HTT exon1. This strategy enables us to rapidly and efficiently replace the RMCE cassette with modified exon1. Hence, we generated a large repertoire of locus-specific genome-edited mESC lines to investigate the functional activities associated with an array of exon1 domains.
Having discovered that the HTT polyQ domain is under purifying selection during evolution (Iennaco et al., Cell Death and Differentiation 2022), we first leveraged the RMCE cell platform to specifically study the functionality of the polyQ tract and found that a stepwise increase of the non-pathogenic polyQ significantly correlates with the neurogenic potential of the cells. We are now more thoroughly investigating the contribution of human and mouse mutant exon1 context to pathogenicity. We found that the human mutant HTT exon1 exhibits more severe phenotypes than its murine counterpart, corroborating the idea that the human HD gene carries increased toxicity. We are now exploiting the RMCE stem cell platform to pinpoint the specific domains/aa in exon1 that are responsible for the observed increased toxicity in humans.

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Mutant huntingtin lowering with ZFPs: neuronal pathophysiology principally drives astrocyte pathology in Huntington’s disease

Baljit S Khakh, PhD, University of California, Los Angeles

Huntington’s disease (HD) is caused by expanded CAG repeats in the huntingtin gene (HTT) resulting in the expression of mutant HTT proteins (mHTT) with extended polyglutamine tracts throughout the brain, including in striatal neurons and astrocytes. However, it is unknown if pathophysiology can be delayed or prevented by lowering mHTT in either cell type throughout the brain, and the relative contributions of neurons and astrocytes to HD pathophysiology remain largely undefined. We used zinc finger protein (ZFP) transcriptional repressors to cell-selectively repress mHTT and evaluated neuronal and astrocytic influences on HD pathophysiology. The major disease drivers were neurons, with astrocytes displaying loss of essential functions such as cholesterol metabolism that were driven by neuronal dysfunction, which encompassed neuromodulation, synaptic, and intracellular signaling. We thus dissected neuronal and astrocytic contributions to a neurodegenerative disease in vivo. Brain wide neuronal ZFPs resulted in strong mHTT lowering and rescue of HD-related behavioral phenotypes. Our data therefore support translational development of ZFPs.

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Altered RNA processing in Huntington’s disease pathogenesis

Jose Lucas, PhD, Center for Molecular Biology Severo Ochoa (CBMSO)

We have analyzed in Huntington’s disease (HD) brains two forms of RNA processing that contribute to post-transcriptional regulation of gene expression: alternative splicing and cytoplasmic polyadenylation.
Alternative splicing generates transcript variants from individual genes, thus increasing molecular diversity. However, when not properly executed it leads to mis-splicing that may result in proteins with altered function and stability. Mis-splicing in three neurodegeneration-linked genes (HTT, MAPT and TAF1) had already been reported in HD. To infer additional splicing alterations relevant to HD pathogenesis, we conducted intersect- RNA-seq analyses of human post-mortem striatal tissue and of R6/1 mice, the latter at early symptomatic stage. Together with a human/mouse parallel motif scan analysis, this approach allowed us to identify the shared mis-splicing signature triggered by the HD mutation in both species and to infer upstream deregulated splicing factors. We are currently dissecting the individual contribution of these splicing factors by generating transgenic mice overexpressing them.
Regarding regulated polyadenylation, cytoplasmic polyadenylation element binding proteins 1 to 4 (CPEB1 to CPEB4) are RNA binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. We have recently reported increased CPEB1 and decreased CPEB4 protein in striatum of HD patients and mice. This correlated with a reprogramming of polyadenylation in 17.3% of the transcriptome, markedly affecting neurodegeneration-associated genes. We found decreased protein content of top deadenylated transcripts, including striatal atrophy–linked genes not previously related to HD, such as KTN1 and the easily druggable SLC19A3 (the ThTr2 thiamine transporter). Mutations in SLC19A3 cause biotin-thiamine–responsive basal ganglia disease (BTBGD), a striatal disorder that can be treated with a combination of the vitamins biotin and thiamine. This led us to discover that, as BTBGD, HD patients show a thiamine deficiency in cerebrospinal fluid (CSF), as well as in brain parenchyma. Currently, we are launching a small open-label clinical trial aimed to discard the potential toxicity associated to the high vitamin doses required to overcome the thiamine deficit induced by decreased ThTr2 expression and exploring, in the Sant Pau (Barcelona) longitudinal cohort, the potential of decreased thiamine as a CSF biomarker of disease progression.

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Molecular mechanisms of HTT degradation

Presentation not posted at presenter’s request

Michael Rapé, PhD, Howard Hughes Medical Institute, University of California, Berkeley

Accumulation and aggregation of HTT is a hallmark of Huntington’s disease (HD). Although carriers of HTT mutations express a polyQ-expanded protein from childhood, phenotypes of HD are only observed later in life. This observation strongly suggests that cells possess quality control pathways that can prevent neurotoxic accumulation and aggregation of HTT, yet the components of this protective pathway have remained unknown. Using a novel imaging platform, we have reconstituted HTT aggregate clearance in neuronal precursor cells, which allowed us to screen for enzymes required for eliminating neurotoxic HTT. This work led to our identification of an E3 ubiquitin ligase that is sufficient to ubiquitylate HTT in vitro and required for HTT aggregate clearance in cells. We propose that hijacking this E3 ligase with small molecules to accelerate HTT ubiquitylation and degradation, i.e. bolstering a natural defense mechanism, will provide a powerful strategy to eliminate HTT in disease.

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Improving gene therapies for HD

Beverly L Davidson, PhD
The Children’s Hospital of Philadelphia & University of Pennsylvania

Gene editing for HD holds much promise, and earlier work by our group and others shows the feasibility of using single nucleotide polymorphisms embedded into PAM motifs residing on the mutant allele to direct allele-specific editing. However, translation to human application requires development on two fronts; i) the timing of expression of the editing machinery (when and for how long), and ii) better methods to deliver the editing machinery to cortical and deep brain structures. To address the first issue we report on our development of a universal switch element, called Xon, for precise control of gene expression. Xon activates gene expression after a single exposure to an orally bioavailable small molecule that, when dosed in humans or mice, can reach both peripheral tissues and the brain. Notably, Xon does not require the co-expression of any regulatory proteins and the robustness of expression can be controlled by the drug dose and the stability of the protein being expressed. Secondly, we will also show our work in advancing delivery modalities for HD based on refinement of AAV vectors for improved brain biodistribution after infusion. These two innovations, in combination, provide both practical and safety advances toward genetic medicines for HD therapy

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RNA-targeting CRISPR/Cas13d system eliminates disease-related phenotypes in preclinical models of Huntington’s disease

Gene Yeo, PhD, MBA, University of California, San Diego

Huntington’s disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Although the pathogenesis of HD remains complex, the CAG-expanded (CAGEX) HTT mRNA and protein ultimately causes disease through a toxic gain-of-function mechanism. As the reduction of pathogenic mutant HTT mRNA is beneficial as a treatment, we developed a CAGEX RNA-eliminating CRISPR-Cas13d system (Cas13d/CAGEX) that binds and eliminates toxic CAGEX RNA in HD patient iPSC-derived striatal neurons. We show that intrastriatal delivery of Cas13d/ CAGEX via a single adeno-associated viral vector (serotype 9) mediates significant and selective reduction of mutant HTT mRNA and protein levels within the striatum of heterozygous zQ175 mice, an established mouse model of HD. Moreover, the reduction of mutant HTT mRNA renders a sustained reversal of HD phenotypes, including improved motor coordination, attenuated striatal atrophy, and reduction of mutant HTT protein aggregates. Importantly, phenotypic improvements were durable for at least 8 months without gross or behavioral adverse effects, and with minimal off-target interactions of Cas13d/CAGEX in the mouse transcriptome. Taken together, we demonstrate a proof-of-principle of an RNA-targeting CRISPR/Cas13d system as a therapeutic approach for HD, a strategy with broad implications for the treatment of other dominantly inherited neurodegenerative disorders

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SHIELD HD – A natural history study to support clinical and biomarker development

Irina Antonijevic, MD, PhD & Peter Bialek, PhD, Triplet Therapeutics, Inc.

Triplet is harnessing the growing evidence that DNA damage response (DDR) genes are potent modifiers of onset and rate of progression of repeat expansion diseases (REDs), such as Huntington’s disease (HD), myotonic dystrophy, and spinocerebellar ataxias. DDR modifier genes act by modulating the rate of somatic expansion at disease-causing gene loci, such as mutant huntingtin (mHTT). The DDR gene MSH3 has been strongly associated with disease progression in HD, including in particular cognitive deterioration that begins in premanifest HD. Only about 50% lowering of MSH3 appears sufficient to halt or markedly slow somatic expansion of mHTT. As no untoward safety signals have been associated with this moderate level of MSH3 lowering, MSH3 has emerged as a prime target with the potential to treat HD and multiple other REDs by acting upstream of individual disease genes.
Triplet is advancing its MSH3-lowering clinical candidate antisense oligonucleotide (ASO) TTX-3360 towards a Phase 1 trial by mid-2022. With a long duration of action in non-human primates (NHPs), TTX-3360 has the potential for infrequent clinical dosing.
To support the Phase 1 trial, Triplet initiated a natural history study, SHIELD HD, in 2020. Premanifest and early manifest people with HD have been enrolled at 9 sites in Europe and North America. Subjects are followed for at least 2 years, with regular clinical, MRI and fluid biomarker assessments. SHIELD HD data allow to 1) perform trial simulations to design a robust Proof-of-Concept (PoC) trial as part of the Phase 1/2a extension, and 2) develop a novel target engagement assay based on measuring MSH3 mRNA in CSF exosomes. In vitro PoC with TTX-3360 was achieved, showing a comparable dose-dependent reduction in MSH3 mRNA in cells and secreted exosomes. Further, MSH3 mRNA was detected in CSF exosomes at similar levels in healthy and premanifest HD subjects. Ongoing analyses of longitudinal studies in NHPs with repeat CSF sampling will inform on the correlation between MSH3 mRNA in CSF exosomes and brain tissues after treatment with TTX-3360. The initial 48-week follow-up data from SHIELD HD and progress on the target engagement biomarker assay will be presented.

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Complement mediates cortico-striatal synapse loss in Huntington’s disease: New biological insight and translation to the clinic

Beth Stevens, PhD, HHMI, Boston Children’s Hospital, Broad Institute

The mechanisms underlying region-specific neuronal degeneration in Huntington’s disease (HD) and how these relate to the development of early cognitive phenotypes are poorly understood. Here, we show that there is selective loss of synaptic connections between the cortex and striatum in postmortem tissue from HD patients that is associated with the increased activation and localization of complement proteins, innate immune molecules, to markers of these synaptic elements. We also find that levels of these secreted innate immune molecules are elevated in the CSF of premanifest HD patients and correlate with established measures of disease burden.
In preclinical genetic models of HD we show that complement proteins mediate the selective elimination of cortico-striatal synapses at an early stage in disease pathogenesis, marking them for removal by microglia, the brain’s resident macrophage population. This process requires mutant huntingtin to be expressed in both cortical and striatal neurons, and inhibition of this complement-dependent elimination mechanism—through either administration of a therapeutically-relevant C1q function-blocking antibody or genetic ablation of a complement receptor on microglia—prevented synapse loss, increased excitatory input to the striatum, and rescued the early development of visual discrimination learning and cognitive flexibility deficits in these models. Together, our findings implicate microglia and the complement cascade in the selective, early degeneration of cortico-striatal synapses and the development of cognitive deficits in presymptomatic HD, and also provide new preclinical data to support complement as a therapeutic target for early intervention.

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Human neural stem cell transplantation for HD

Leslie M Thompson, PhD, University of California, Irvine

Huntington’s disease (HD) most overtly impacts the striatum, with progressive loss of medium spiny neurons and atrophy of the cortex. At a molecular level, the disease is accompanied by a progressive loss of neuronal proteins, including the neurotrophic factor BDNF that supports the survival of striatal neurons. Further, aberrant accumulation of aggregated huntingtin (HTT) protein species corresponds to disease pathogenesis. Stem cell-based approaches are promising as a treatment option for HD with potential to modulate pathology in complex tissues such as the brain. We have developed and extensively evaluated a GMP-compliant human embryonic stem cell (ESC)-derived neural stem cell (NSC) product, ESI-017 hNSCs, for transplantation into the striatum of HD patients, with the intent of slowing or preventing the progression of the disease. The transplanted ESI-017 hNSCs have been tested in mouse models of HD. In both R6/2 and zQ175 mice, cells engraft and differentiate to neuronal populations, express BDNF and reduce mutant HTT accumulation. These molecular and histological improvements correlate with behavioral improvement in HD mice. Transplantation of human NSCs is challenged by the need for long-term functional integration. Following long-term transplantation in zQ175 mice, host tissue appears to form synaptic contacts with transplanted cells as evidenced by EM, suggesting they may provide new circuitry to reduce the aberrant cortical excitability that occurs in human HD. Following 8-month implantation of hNSCs into the striatum of zQ175 HD mice, patch clamp recordings, immunohistochemistry and electron microscopy demonstrates that hNSCs differentiate into diverse neuronal populations. hNSCs appear to receive synaptic inputs, innervate host neurons, and improve membrane and synaptic properties. Single nuc RNAseq on transplanted tissues following 6 months transplantation indicates three major populations of cells. Overall, the findings support hNSC transplantation for further evaluation and clinical development for HD. IND enabling safety studies in preclinical models of HD are currently ongoing.

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The coming of age of de novo protein design

David Baker, PhD, University of Washington

Proteins mediate the critical processes of life and beautifully solve the challenges faced during the evolution of modern organisms. Our goal is to design a new generation of proteins that address current-day problems not faced during evolution. In contrast to traditional protein engineering efforts, which have focused on modifying naturally occurring proteins, we design new proteins from scratch based on Anfinsen’s principle that proteins fold to their global free energy minimum. We compute amino acid sequences predicted to fold into proteins with new structures and functions, produce synthetic genes encoding these sequences, and characterize them experimentally. In this talk, I will describe the de novo design of SARS-CoV-2 candidate therapeutics, synthetic antagonists and agonists of cellular receptors, molecular machines, and recent advances in deep learning-based structure modeling and design.

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Biomarker Task Force: A collaboration to enable the community

Cristina Sampaio, MD, PhD & Robert E Pacifici, PhD, CHDI

Biomarkers have become invaluable tools in oncology clinical trials and are now acquiring critical importance in neurodegenerative drug development programs, where early signals of safety and efficacy can improve quality, cycle-time, and cost. CHDI has assembled a Biomarker Task Force to conduct a careful ‘gap analysis’ assessment of the unmet needs for the existing and planned HD drug development programs, leading us to identify four near-term research goals: additional pharmacodynamic biomarkers for huntingtin lowering; pharmacodynamic biomarkers for somatic instability; on-mechanism safety biomarkers for huntingtin
lowering; and biomarkers that are diagnostic of disease stage, with an emphasis on early stages. We have developed a portfolio of strategies and experimental approaches to achieve these goals and implementation is now underway, and these activities and plans will be presented. Biomarkers can be as difficult to develop as the candidate therapies and we hope that the wider HD community will join our vision for a precompetitive initiative that seeks to build synergies, reduce redundancy, and benefit us all.

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Multi-omic profiling of people with Huntington’s disease (PwHD) for biomarker discovery: The future is bright!

Jim Rosinski, PhD, CHDI

There is a critical need in HD research for accurate and reliable methods to track individual disease progression stages and response to candidate therapeutics. As part of CHDI’s drive to identify clinically-ready biomarkers, we implemented a strategy to build new and leverage existing large clinical omics discovery datasets to analyze and advance potential candidates as disease-monitoring or treatment-response biomarkers. An overview of our current strategy across DNA epigenetics and RNA, proteomic, and metabolite profiling will be presented, highlighting our proteomics studies and recent results from the HDClarity study. An initial study on PwHD and control samples from 579 matching CSF and serum samples have been profiled on the SomaLogic SomaScan platform (>7000 protein analyte levels) and, from our initial analysis, candidate biomarkers correlate to CAP score and clinical endpoints (e.g., cUHDRS); several protein analytes not previously nominated as candidates also merit further evaluation. We have also begun to evaluate multiprotein signatures as potential predictors of HD stage and clinical outcomes. The SomaScan proteomics platform is being used in parallel with discovery mass spectrometry-based proteomics in collaboration with Niels Skotte and Matthias Mann, along with evaluation of additional proteomics platforms (such as Olink). Our overall strategy is to expand the evaluations in HDClarity/Enroll-HD to develop multi-omics datasets across DNA, RNA, protein, and metabolites to enable a multimodal data integration incorporating imaging, digital, and clinical phenotypes with molecular measurements.

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Biodistribution, dosimetry, and first-in-human evaluation of 11C-CHDI-180R and 11C-CHDI-626 as radioligands for cerebral mutant huntingtin PET imaging

Aline Delva, MD, KU Leuven

Clinical development of mutant huntingtin (mHTT)-lowering therapies would benefit from techniques that can reliably quantify mHTT levels in the human brain. 11C-CHDI 00485180 R (11C-CHDI 180R) and 11C-CHDI 00485626 (11C-CHDI 626) are two novel PET radioligands targeting mHTT. Biodistribution, dosimetry, and kinetic properties of both radioligands in healthy controls (HCs) will be presented. As a brain-penetrable metabolite was formed for 11C-CHDI 626, only 11C-CHDI-180R was suitable for further exploration. Additionally, the results of a phase 1 study to investigate brain quantification of 11C-CHDI 180R in HD patients and HCs will be disclosed. These data support that 11C-CHDI 180R is a promising PET radioligand targeting mHTT.
Synaptic damage has long been suspected to play a major role in the pathophysiology of HD, but in vivo evidence in humans is limited. To assess synaptic damage and its clinical correlates in early HD, we performed a PET imaging study with 11C-UCB-J, a radioligand for the ubiquitous presynaptic terminal marker SV2A, and 18F-FDG, as regional cerebral glucose consumption is thought to largely reflect synaptic activity. 11C-UCB-J PET revealed extensive loss of SV2A in early HD, suggesting widespread synaptic disconnection. SV2A loss in the putamen correlated with motor impairment. 11C-UCB-J PET was shown to be more sensitive than 18F-FDG PET to detect extrastriatal changes in early HD.

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Digital monitoring of cognitive and motor symptoms in Huntington’s disease: preliminary longitudinal findings from the GENERATION HD1 study of tominersen

Peter McColgan, MD, PhD & Jonas Dorn, PhD, Roche Products Ltd & Roche Pharma Research and Early Development

Roche responds to additional post-presentation questions

Continuous remote monitoring of Huntington’s disease (HD) signs and symptoms has the potential to complement standard in-clinic tests that may be limited by inter-rater variability, subjectivity and naturally occurring fluctuations in symptom intensity. The Roche HD digital monitoring platform (dMP), consisting of smartphone-based active tests as well as smartphone- and smartwatch-based passive monitoring, has been shown to have good test-retest reliability, cross-sectional validity and cross-sectional correlations with standard clinical measures of cognitive and motor performance.
GENERATION HD1 (NCT03761849) is a randomised, double-blind, placebo-controlled study in 791 patients with manifest HD assessing 120 mg tominersen administered every 8 (Q8W) or 16 weeks (Q16W). In March 2021, GENERATION HD1 dosing stopped following an independent data monitoring committee recommendation: at Week 69, the Q8W regimen showed an unfavourable benefit:risk profile versus placebo; the Q16W regimen showed no benefit and a similar safety profile to placebo.
Exploratory digital assessments using the dMP in GENERATION HD1 include: Symbol Digit Modalities Test; Stroop Word Reading; Speeded Tapping Test; Draw-a-Shape Test; Chorea Test; Balance Test; U-turn Test; Walk Test; questionnaires such as EQ-5D-5L (a self-assessed quality of life questionnaire); and passive monitoring. Patients were provided with a smartphone and smartwatch, and trained on their use during screening. Patients were asked to perform active tests daily at home according to a predefined schedule. For passive monitoring, patients were asked to carry the devices with them as they went about their daily activities.
Preliminary findings from GENERATION HD1 based on data from the dMP up to Week 69, prior to stopping dosing, will be presented.
GENERATION HD1 was sponsored by F. Hoffmann-La Roche Ltd.

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Huntington’s Disease Integrated Staging System (HD-ISS): A primer for use in clinical research

Sarah J Tabrizi, MD, FRCP, PhD, FMedSci & Jeffrey D Long, PhD

The HD Integrated Staging System (HD-ISS) comprises a biological research definition of HD and evidence-based staging centered on prognostic biological, clinical, and functional landmarks. It is the result of a formal consensus process within the Huntington’s Disease Regulatory Science Consortium’s Regulatory Science Forum (RSF), a working group made up of expert representatives from industry and academia. The RSF considered prognostic biomarkers, signs, and symptoms of HD, and performed empirical data analysis. It used observational data to calculate healthy-control-based landmark variable cut-offs for stage classification and to internally validate the framework.
The HD-ISS starts with Stage 0, which comprises individuals with ≥ 40 CAG repeats in the huntingtin gene (HTT) before detectable indications of disease pathology. HD progression is verified by assessments of underlying pathophysiology (Stage 1), then proceeds to a detectable clinical phenotype (Stage 2) and continues to a decline in function (Stage 3). Operationally, individuals can be classified into Stages 1-3 based on CAG-independent thresholds of landmark assessments. Importantly, the HD-ISS covers the entire disease spectrum starting from birth, and is unconstrained by concepts such as “manifest,” “premanifest,” or “prodromal.”
This presentation additionally gives two practical applications of the HD-ISS. First, it demonstrates the use of the “calculator” website, which uses inputs of the landmark assessments for a given individual to calculate the HD-ISS stage for that person. Second, it gives some examples of why, and how, to enrich within the HD-ISS stages for clinical trial design.
The HD-ISS allows the definition of study populations across the entire duration of the disease trajectory, including before clinical motor diagnosis. Clinical trials using the HD-ISS can be implemented immediately. In HD-ISS Stages 2 and 3, we have knowledge of how to enrich and what endpoints to measure. Although clinical trials in Stage 1 may require further research, that work is ongoing. While participant populations may also be defined by additional enrichment criteria as determined by the study objectives, we propose that all new clinical studies use the HD-ISS.
Co-presenters: Emily C Gantman, PhD, Alexandra Mansbach, JD, & Cristina Sampaio, MD, PhD

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Understanding the treatment and off-treatment effects of tominersen in the Phase III GENERATION HD1 study

Peter McColgan, MD, PhD & Lauren Boak, PhD
Roche Products Ltd & F. Hoffmann-La Roche Ltd

Roche responds to additional post-presentation questions

Tominersen is an antisense oligonucleotide lowering huntingtin protein. GENERATION HD1 (NCT03761849) is a randomised, double-blind, placebo-controlled study in 791 patients with manifest Huntington’s disease (HD) assessing 120 mg tominersen administered every 8 (Q8W) or 16 weeks (Q16W). In March 2021, GENERATION HD1 dosing stopped following an independent data monitoring committee (iDMC) recommendation: at Week 69, Q8W showed an unfavourable benefit:risk profile versus placebo; Q16W showed no benefit and a similar safety profile to placebo.
Analyses of the GENERATION HD1 off-treatment period showed the difference in slope of decline in clinical endpoints in Q8W/Q16W was not statistically significant compared with placebo. Group averaged ventricular volumes, cerebrospinal (CSF) protein and leukocyte cell count showed improvement compared with the on-treatment period. The iDMC reviewed available data and concluded no additional follow-up is required.
GENERATION HD1 failed to meet its original objectives. Hypothesising that age, disease burden (measured by CAG age-product [CAP] score) and/or drug exposure possibly contributed to outcomes, post hoc analyses for each dosing regimen were conducted using a mixed model for repeated measures through Week 69 in subgroups (defined by median value at baseline prior to unblinding): CAP <500/≥500; age <48/≥48 years. The Q16W low-age/low-CAP subgroup was further split based on median CSF average exposure of tominersen at Week 21 to further evaluate the impact of exposure.
In the Q16W low-age/low-CAP subgroup, point estimates for Unified HD Rating Scale (UHDRS) clinical endpoints at Week 69 were in the favourable direction versus placebo. Qualitative sensitivity analyses across age and CAP support these findings. Other subgroups were generally unfavourable or comparable with placebo.
When the Q16W low-age/low-CAP subgroup was split by exposure, point estimates in the low-exposure group were generally in the direction of improvement and point estimates in the high-exposure group were comparable to placebo.
Our findings support the continued development of tominersen in a prospectively designed, randomised, placebo-controlled study in younger patients with less disease burden. Considerations for dose regimen and patient population selection in the new study will be discussed.

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A prospective pilot trial for pallidal deep brain stimulation in Huntington’s disease

Jan Vesper, MD, PhD, Heinrich Heine University Duesseldorf

Background: Huntington´s disease is often medically refractive. Case reports suggest deep brain stimulation as a treatment option, but the exact target for stimulation is under debate. We previously assessed procedure safety, equality of internal and external pallidal stimulation in patients with HD followed up for 6 months. We hereby report on the first prospective, randomised, double blind, parallel group, sham-controlled, multi-centre superiority trial.
Methods: Surgery was performed under general anaesthesia, including bilateral stereotactic insertion of quadripolar electrodes (Medtronic Inc., 3387) into the GP and stimulator implantation (ACTIVA® PC) for chronic stimulation. A 1:1 randomization followed one week after surgery (12 weeks parallel group, 12 weeks open follow-up). The aim of the study was to prove the efficacy and safety of pallidal DBS in HD patients and to show superiority of DBS on motor function in the stimulation group compared to stimulation-off group. Difference between the groups were evaluated in the UHDRS total motor score (UHDRS-TMS) at 12 weeks (primary endpoint) postoperatively compared to baseline.
Results: 8 European sites participated, 44 patients were randomized out of 80 patients who have been assessed for eligibility. Finally, 40 were eligible for final analysis (4 drop outs). Extensive clinical evaluation was performed at baseline, 3 and 6 months postoperative follow-up visit: motor function; Unified Huntington’s Disease Rating Scale (UHDRS) chorea subscore, bradykinesia subscore, Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) motor score, Q-Motor “choreomotography” and “digitomotography” test; cognition; Mattis Dementia Rating Scale (MDRS), Verbal Fluency Test, Symbol Digit Modalities Test (SDMT), STROOP word reading, color naming and color of the word naming; psychiatry/emotion; Hospital Anxiety and Depression Scale combined (HADS-SIS), Problem Behaviours Assessment Short Form (PBA-s), Quality of Life: SF-36, Clinical Global Impression Scale (CGI). All adverse events resolved without sequelae. No unanticipated AEs and SAEs occurred, no ICH. 1 patient died during open phase (after 12 weeks). DSMB determined this event as unrelated to surgery. 1 infection occurred 12 weeks after surgery.
Discussion: DBS in HD was found to be safe treatment option for reduction of chorea in HD. Preliminary clinical data will be presented during the conference. Further evaluations are ongoing, including independent video rating of clinical results as a secondary endpoint.

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Innovations that led to SELECT-HD, a phase1b/2a clinical trial of an allele-selective therapy for Huntington’s disease

Michael A Panzara, MD, MPH, Wave Life Sciences

In September 2021, Wave Life Sciences initiated a phase 1b/2a clinical trial (SELECT-HD) of the investigational stereopure oligonucleotide, WVE-003, in patients with early manifest Huntington’s disease (HD). WVE-003 targets a single nucleotide polymorphism (SNP3) that is associated with the mutant allele of the huntingtin gene (mHTT). This approach is intended to enable allele-selective lowering of mHTT and relative sparing of wild-type HTT (wtHTT). Preservation of wtHTT may provide a benefit over non-selective mHTT-lowering, as wtHTT protects neurons from cellular stress, supports neuronal circuits, and promotes the clearance of brain catabolites.
We will discuss the innovations that underpin SELECT-HD. WVE-003 incorporates new chemistries that have been shown to improve the pharmacological properties of oligonucleotides in preclinical studies, both in terms of potency and distribution to key brain regions, supporting intrathecal delivery. To enable rapid patient identification for an allele-selective therapy, Wave and Asuragen developed an investigational assay that enables SNP genotyping and phasing with the huntingtin gene. To evaluate the allele-selective mechanism of WVE-003, Wave developed a new method for reliable quantification of the wtHTT protein in human CSF. We will provide insight into the development, optimization, and fit for purpose qualification of this wtHTT protein quantification method. And finally, we will review SELECT-HD’s adaptive design. Adaptive trial designs have proven successful in oncology but have not yet been widely implemented in neurology. By designing SELECT-HD to be adaptive, Wave can make data-driven changes to dose level and dosing frequency while the trial is ongoing. As a result, we believe an adaptive design will expedite the evaluation of WVE-003.

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HD-GeneTRX-1 and HD-GeneTRX-2: Phase 1/2 clinical trials of AMT-130 gene therapy for early-stage HD

David L Cooper, MD, MBA, uniQure

Huntington’s disease (HD) is a gradually progressing, irreversible, neurodegenerative disorder leading to disability and death, with no disease modifying therapies currently available to delay onset or to slow disease progression. uniQure is conducting a phase I/II clinical trial (HD-GeneTRX1; NCT04120493) of AMT-130 (AAV5-miHTT), which is an AAV-based gene therapy designed to reduce total huntingtin protein. This randomized trial is exploring the safety, tolerability, and efficacy in early manifest HD patients with 5 years total follow-up (12 months blinded). A total of 26 patients will be randomized for treatment or sham (control) with 6 patients in the low-dose cohort, (6×1012 vg), 10 patients in the high-dose cohort (6×1013 vg), and 10 patients in the sham surgery arm (eligible for crossover). Patients in the treatment arms will receive a single administration of AMT-130 through MRI-guided, convection-enhanced stereotactic neurosurgical delivery directly into the striatum (caudate and putamen). The experience in this first-in-human blinded trial of intraparenchymal administration of gene therapy in patients with HD has informed many updates to the trial design, including modifications of the inclusion/exclusion criteria to optimize screening for patients with early manifest motor or multidimensional HD who have sufficient striatal volume. More recently, a European, open-label phase Ib/II study (HD-GeneTRX2; EudraCT 2020-001461-36) of AMT-130 was initiated that will enroll 15 patients with early manifest HD across two dose cohorts with parallel clinic visits and assessments to further establish safety, proof of concept, and the optimal dose of AMT-130. Based upon the experience with the administration procedure, uniQure also plans to initiate a third blinded cohort in the US study of up to 18 additional randomized patients (6×1013 vg) that will explore the use of alternative stereotactic navigation systems to simplify placement of catheters for the MRI-guided convection enhanced infusions as a further bridge towards phase III.

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PTC518: Clinical development update

Brian Beers, BSc, PTC Therapeutics, Inc

Huntingtin (HTT) lowering has significant therapeutic potential for Huntington’s disease (HD), which is caused by an autosomal dominant polyglutamine repeat expansion mutation in the HTT gene. No disease-modifying treatments are currently available. Multiple huntingtin-lowering strategies are being employed in clinical and preclinical studies for the treatment of HD, including antisense oligonucleotides, RNA interference, zinc-finger transcriptional repressors and CRISPR/Cas9 gene editing. Our objective is to address the unmet medical need for patients with HD by developing orally bioavailable HTT-lowering molecules that would delay the onset or slow the progression of their disease. The HTT-lowering program is part of PTC’s proprietary pre-mRNA splicing platform, where we leverage our knowledge of splicing regulation as a novel platform to discover and develop small-molecule splicing modifiers. Utilizing this cutting-edge technology, we have identified small molecule splicing modifiers that cross the blood brain barrier upon oral delivery, and uniformly lower HTT levels in the key affected areas of the HD mouse brain – the striatum and cortex. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels
Here we present the results from a first-in-human, Phase I trial of PTC518, an HTT-lowering small molecule which was identified as part of PTC’s splicing drug-discovery platform, as well as the proposed design for a Phase II clinical trial of PTC518 in patients with HD.

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VIBRANT-HD: A phase 2b trial to investigate the safety, tolerability, pharmacodynamics and pharmacokinetics of branaplam, an oral HTT lowering molecule, in people with early manifest HD

Beth Borowsky, PhD, Novartis Pharmaceuticals

Branaplam is an orally available small molecule mRNA splice modulator that promotes the inclusion of a pseudoexon in HTT mRNA, leading to a reduction in levels of normal and mutant HTT mRNA and protein. Following oral dosing, branaplam gets to key brain regions to lower mHTT levels in HD mouse models. Chronic branaplam lowers plasma HTT mRNA levels in SMA children, and a single dose lowers HTT mRNA and protein in healthy adults. Branaplam is now being investigated in a Ph2b trial, VIBRANT-HD, in which people with early manifest HD are randomized to either placebo or one of three different doses. All participants completing the initial dose-finding part will then be eligible to enter an open-label extension and receive the dose of branaplam that is selected for further development. Compared to HTT lowering therapies delivered intrathecally or intracranially, an oral HTT lowering therapy should be less burdensome to patients, should lead to a more uniform lowering of mHTT throughout the brain, and should be quickly reversible.

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