CHDI FOUNDATIONCHDI

Accelerating Therapeutic Development for Huntington's Disease

2023 Conference

CHDI’s 18th Annual HD Therapeutics Conference took place April 24 – 27, 2023, in Dubrovnik, Croatia, to allow more Europe-based colleagues to attend. This unique conference series focuses on drug discovery and development for Huntington’s disease, drawing 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.

Fundamental outstanding questions in HD therapeutic development

Vahri Beaumont, PhD, CHDI

The discovery in 1993 that Huntington’s disease is caused by an expanded (CAG)n repeat within the huntingtin (HTT) gene was a profound catalyst for the field, dramatically accelerating research and raising hope that successful therapeutic strategies would soon follow. Thirty years on, much has been accomplished: HTT-lowering trials are well underway, a global registry enabling recruitment of well-characterized clinical trial participants is established, more refined clinical outcome scales are under development, and biomarkers of disease progression and HTT lowering have been and are being developed. Further, additional genetically validated targets to modify disease trajectory have been identified. It is a thrilling time for HD research, but it is humbling that clinical success has yet to be achieved.
In this perspective I will discuss some of the advancements and disruptive technologies that have led to our ongoing redefinition of our understanding of HD. I will explore the fundamental questions that remain to be answered, such as the precise molecular and pathophysiological bases of cell vulnerability/resilience, and the spatiotemporal sequence of CNS dysfunction giving rise to HD clinical signs and symptoms. As a prelude to the scientific sessions to follow, I will underscore how these insights are critical to enable us to develop therapeutics with the correct target product profile. These range from our knowledge of the targets themselves, associated safety concerns, the modality employed, the correct temporal application and the optimal biodistribution.

Top

Understanding the contribution of genetic variation in HTT to Huntington’s disease and other traits in humans

Galen Wright, PhD, University of Manitoba

This year marks the 30th anniversary of the discovery of the pathogenic role of the HTT gene in Huntington’s disease (HD). These initial studies made significant progress in showing that the length of the expanded CAG repeat in the first exon of the gene was inversely correlated with the age of onset of HD. However, HD age-of-onset prediction models only explain a subset of the variability observed in the clinic. Recent advancements in human genomics have allowed the field to tackle these questions in an unprecedented manner.
For example, large-scale genomic studies have highlighted DNA repair genes and somatic repeat instability in modifying the age of clinical onset in HD in large cohorts of affected individuals. Further, the recent discovery that interrupting sequence variants within the HTT CAG tract dramatically impact HD age of onset has changed our understanding of HD biology. These variants do not alter the protein sequence and only change the underlying DNA sequence, yet they make a significant impact on clinical outcomes. The current presentation will cover the biological and clinical implications of these recent findings, including diagnostic testing considerations, pertinent populations (i.e., reduced penetrance allele carriers, CAG 36-39) and new therapeutic targets. Currently unanswered questions regarding somatic repeat instability and HTT CAG interrupting sequence variants will also be discussed.
Finally, human genomics advances are beginning to identify new associations between genetic variants in the HTT gene and human disease-relevant traits. This includes Lopes-Maciel-Rodan syndrome, caused by compound heterozygous mutations in HTT. This presentation will highlight key findings from our recent analysis of common huntingtin-related genetic variation using an unbiased analysis of over 50,000 human genetic studies combined with machine learning-based fine mapping. These results underscore the beneficial neurogenic role of HTT in modifying neurobiological traits outside of HD, pointing to roles for the gene in aging. (e.g., parental lifespan, longevity, frailty index). We are extending this analysis pipeline to other polyglutamine disorder genes to delineate further the underlying biological processes these genes participate in and further understand HTT/HD biology.

Top

Crosstalk between DNA repair systems in the CAG-repeat expansion in Huntington’s disease

Anna Pluciennik, PhD, Thomas Jefferson University

The CAG-repeat expansion within the huntingtin (HTT) gene is the causative mutation of Huntington’s disease (HD), a progressive neurodegenerative disorder characterized by loss of medium spiny neurons in the striatum. The inheritance of the CAG repeat at the HTT locus is sometimes characterized by changes in repeat length, and individuals who inherit longer repeats develop the disease at an earlier age. HD patients also display tissue-specific somatic CAG expansion, with a high degree of age-dependent CAG expansion observed in vulnerable tissues such as the striatum and cortex. However, the exact molecular mechanisms of CAG-repeat expansion is not known. Human genome-wide association studies have identified FAN1 and several DNA mismatch repair (MMR) genes as modifiers of HD age of onset. In animal models, FAN1 prevents somatic expansion of CAG triplet repeats whereas MMR proteins promote this process. To understand the molecular basis of these opposing effects, we evaluated FAN1 nuclease function on DNA extrahelical extrusions that represent key intermediates in triplet-repeat expansion. Our studies describe a strand-directed, extrusion-provoked nuclease function of FAN1 that is activated by RFC, PCNA, and ATP at physiological ionic strength. Activation of FAN1 in this manner results in DNA cleavage in the vicinity of triplet repeat extrahelical extrusions thereby leading to their removal in human cell extracts. The role of PCNA and RFC is to confer strand directionality to the FAN1 nuclease, and this reaction requires a physical interaction between PCNA and FAN1. We also show that FAN1-dependent CAG extrusion removal relies on a very short patch excision-repair mechanism that competes with MutSβ-dependent MMR (a process characterized by longer excision tracts). These results provide a mechanistic basis for the role of FAN1 in preventing repeat expansion and could explain the antagonistic effects of MMR and FAN1 in disease onset/progression. We propose that the balance between these opposing pathways likely determines the rate of repeat expansion and may be critical for maintenance of genomic stability.

Top

The role of R-loops in HD pathogenesis

Natalia Gromak, PhD, University of Oxford

Huntington’s disease (HD) is one of 50 repeat expansion diseases where a CAG-triplet expansion in the first exon 1 of the huntingtin (HTT) gene leads to production of a mutant HTT protein. HD is associated with a plethora of cellular pathological events, leading to a striatum degeneration in HD patients. Despite well-defined genetic cause, the molecular mechanisms underlying HD pathology are still not fully understood. The evidence from multiple triplet-expansion diseases suggests that unusual nucleic acid structures are involved in their pathogenesis. R-loops are non-canonical structures composed of an RNA/DNA hybrid and a single stand of DNA formed during transcription. Recently, R-loops have emerged as key regulators of important biological processes including transcription, DNA replication, generation of antibody diversity and epigenetics. However, if not properly regulated, R-loops can become pathogenic and accumulate in neurodegenerative disorders and cancer. We showed that R-loops form over the expanded repeats in Friedreich’s ataxia and fragile X syndrome, where they associate with formation of repressive chromatin marks and gene silencing, leading to the disease pathology (Groh et al. 2014). Until now, the contribution of R-loops to HD pathology has not been investigated. Our lab has developed multiple tools in the R-loop field which we are currently employing to study the role of these structures in HD. I will present preliminary data investigating a potential role of R-loops in pathology using different HD models. Ongoing work is focused on understanding the role of R-loops in repeat instability and genome regulation. Collectively, our data provide novel insights towards HD pathogenesis, potentially uncovering new targets for therapeutic interventions.

Top

In vivo gene editing of the mutant huntingtin gene

Michael Brodsky, PhD, UMass Chan Medical School

CRISPR-Cas based gene-editing technologies offer an exciting therapeutic opportunity for the treatment of genetic disorders such as Huntington’s disease (HD). Ideally, a single treatment would permanently alter the structure of the mutant HTT gene, preventing expression of the encoded toxic RNAs and proteins. However, several aspects of HD present challenges for gene editing. Editing should leave one normal copy of HTT intact. In addition, the editing mechanism must work in post-mitotic cells such as neurons, which only utilize a subset of DNA repair pathways. We have developed two gene-editing approaches that address these concerns. The first approach takes advantage of previously described SNP heterozygosities present in the HTT gene of a subset of HD patients. Expression of CRISPR-Cas9 nucleases targeting coding region SNPs induces allele-specific inactivation of mHTT in the striatum of a Hu97/18 mouse model. The second approach takes advantage of the inherent instability of expanded CAG repeats. Expression of dual nickases that specifically target the CAG repeats in HTT exon 1 can partly correct mHTT mutation contraction of the CAG-repeat tract. This method can reduce mHTT CAG repeat numbers in both HD patient-derived fibroblasts and in the striatum of the mouse Q50 knockin model. The strengths and limitations of these two approaches will be discussed.

Top

Genome-editing approaches to treat Huntington’s disease

Ben Kleinstiver, PhD, Massachusetts General Hospital & Harvard Medical School

Genome editing approaches to treat certain diseases are complicated by the sequence complexity of the pathogenic mutation. For instance, expanded DNA repeats that cause a range of disorders, such as Huntington’s disease, are difficult to target efficiently and safely. Two main approaches to edit expanded repeat alleles have been pursued, including direct perturbation of the repeat itself or targeting flanking haplotype-associated SNPs to knockout the expanded allele. However, these approaches pose challenges for conventional genome editing technologies due to an inability to precisely and accurately target the repeat or flanking SNPs, unpredictable edit outcomes, or low overall editing efficiency. Thus, there is an opportunity to overcome key challenges of editing HTT and other genes that cause repeat-expansion diseases, by leveraging protein engineering approaches to improve the inherent properties of CRISPR-Cas technologies. To circumvent the limitation of protospacer-adjacent motifs (PAMs) that preclude precise user-specifiable targeting of DNA bases with CRISPR enzymes, we’ve undertaken a range of approaches to reduce or alter the PAM requirement. For instance, our recently developed Cas enzymes that require minimal PAMs offer unparalleled access to the genome, and together enable CAG-repeat targeting with single nucleotide resolution1-3. In an alternate approach, we have also engineered a series of CRISPR enzymes with more stringent PAM requirements4,5, which permit safe and efficient allele-specific targeting of repeat-associated SNPs. We are deploying this catalog of engineered CRISPR enzymes in various formats (as nickases, nucleases, base editors, prime editors, etc.) to explore which approach leads to maximal HTT gene editing with minimal unwanted edits. Together, these engineered enzymes offer new capabilities for targeting and perturbing the genetic causes of disorders caused by expanded DNA repeats, providing tools to study and potentially treat this class of devastating diseases.
1 Kleinstiver et al., Nature Biotechnology, 2019
2 Walton et al., Science, 2020
3 Christie et al., Nature Biotechnology, 2022
4 Kleinstiver et al. Nature, 2015
5 Kleinstiver et al., Nature Biotechnology, 2015

Top

Allele-selective SNP editing utilizing AAV5-delivered LETI nuclease and guide RNA resulting in meaningful reduction of mutant HTT protein

Presentation not made available by presenter

Kathryn Woodburn, PhD, Life Edit Therapeutics

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene. The resulting polyglutamine-containing mutant HTT (mutHTT) disrupts wild-type HTT (wtHTT) function resulting in neural stress and malfunction. Murine models containing expanded CAG repeats recapitulate HD pathophysiology. Reducing mutHTT levels in HD animal models result in amelioration of motor and neuropathological abnormalities, supporting HTT-lowering as a therapeutic approach. wtHTT has been shown to support critical neural function, thus the selective strategy targeting only the disease-associated mutHTT is favored.
The enablement of a single-nucleotide polymorphism (SNP) based allele-selective strategy in lowering mutHTT was evaluated in a clinically relevant murine HD model. Life Edit Gene (LEG) nuclease and concordant single guide RNA (SGN), targeting a critical SNP, were packaged in adeno-associated virus (AAV) serotype 5 and administered bilaterally in HD murine striatum. Subsets of mice were evaluated at 4 weeks and 3 months post administration and compared to concurrent control animals. Clinical observations, human mutHTT mRNA and protein levels, mRNA and protein LEG levels, AAV5 distribution and editing efficiency (NGS) were evaluated.
Administration of SGN-LEG-AAV was well tolerated. AAV5 delivery via intrastriatal administration resulted in high vector genome copy number in both striatum and cortex. A temporal and dose response reduction in mutHTT was observed with clinically meaningful reductions >40% observed. Percent INDELs correlated with reduction in mutHTT levels. The results of these studies support the continued development of SGN-LEG-AAV for potential allele-specific reduction of mutHTT.

Top

Understanding MutSβ structure, function, and modulation by small molecules

Dan Felsenfeld, PhD and Tasir Haque, PhD, CHDI

Human genome-wide association studies have identified several genes in the DNA mismatch repair (MMR) pathway as modulators for the age at onset and progression of Huntington’s disease (HD). CHDI has been investigating approaches to exploiting the possible link between MMR machinery and somatic expansion for therapeutic benefit in the treatment of HD. With a focus on modulating the activity of the MMR protein complex MutSβ—a heterodimeric DNA-binding ATPase comprised of the MSH2 and MSH3 subunits—ongoing work has sought to determine the structure of the protein complex, developing biochemical and cellular assays for MMR activity, and identifying small-molecule tool compounds that modulate this pathway. Significant progress has been made in elucidating both the function and structure of the MutSβ complex, including cryo-electron microscopy structures in both the presence and absence of a DNA substrate and X-ray crystal structures of small-molecule ATPase inhibitors bound to MutSb. Additionally, inhibition of the protein-protein interaction between MutSβ and MutLα is also being evaluated as another approach to modulating MMR activity. These new assays and structures will advance the scientific community’s understanding of MMR and MutSβ and aid in developing tool molecules, paving the way to new opportunities for therapeutic intervention.

Top

Drugging mismatch repair pathways for Huntington’s disease

Caroline L Benn, PhD, LoQus23 Therapeutics

The causative mutation for HD, an expanded trinucleotide repeat sequence in the first exon of HTT, is naturally polymorphic and indisputably associated with disease phenotypes. Indeed, CAG-repeat length is the main driver for disease onset and progression, with longer repeats associated with worsening prognosis. We also know that CAG repeats are somatically and intergenerationally unstable, with a bias toward expansion in a length-, time-, and tissue-dependent fashion. Genome-wide association studies in HD and other trinucleotide repeat expansion diseases have highlighted genes involved in mismatch repair (MMR) pathways as modifiers for clinically relevant HD symptomatology, including age at onset of motor and cognitive symptoms, rate of disease progression, and somatic instability. Furthermore, repeat stability (or lack of) has been shown experimentally to be mediated by proteins that function in MMR processes. This raises the tantalizing potential for a novel disease modifying therapeutic: by modulating the function of such MMR proteins, could one significantly delay the onset of disease symptoms and slow the rate of progression through prevention of somatic expansion and increased mutational burden over time?

Top

Inhibition of MutSβ activity as a potential treatment for Huntington’s disease

Presentation not made available by presenter

James Fleming, PhD, Pfizer Inc.

Genetic modifiers of age of onset in Huntington’s disease (HD) provide compelling evidence that somatic expansion of the CAG repeats is a critical driver of pathogenesis, and data from mice and humans demonstrate that the DNA mismatch repair (MMR) pathway is crucial for repeat instability. Due to the apparent sensitivity of repeat expansion to MutSβ levels and the limited association with cancer, we evaluated a subunit of MutSβ, MSH3, as a potential target for small-molecule therapeutic intervention. We established a cellular assay for detecting repeat instability and demonstrated that mutations in the ATPase domain of MSH3 mimic the effect of an MSH3 knockout. Having validated the MSH3 ATPase domain as a therapeutic target, we conducted a high-throughput screen and identified multiple scaffolds that competitively inhibit MutSβ ATPase activity. Native mass spectroscopy confirmed compound binding to MutSβ, and biophysical studies demonstrate that these compounds modulate the binding kinetics of MutSβ to DNA containing a loop out. Furthermore, we demonstrate compounds inhibit the downstream interaction with MutL, an essential step in MMR. Crystallography revealed selective binding of compound at the MSH3 ATPase pocket, and structure-guided optimization of the lead series yielded compounds with nanomolar potency and excellent selectivity versus MutSα (>100-fold). Critically, we demonstrate that exemplars in our compound series modulate MutSβ activity in cellular assays that measure both DNA loop repair as well as repeat instability. Current efforts are focused on developing molecules that maintain potency against MutSβ while improving physiochemical and pharmacokinetic properties that will enable us to demonstrate pharmacological modulation of CAG-repeat instability in HD mouse models. This work demonstrates that targeting the ATPase activity of MutSβ with small molecules is a promising therapeutic approach for modulating somatic instability in HD.

Top

Parsing pathogenesis: Instability and toxicity

Jim Gusella, PhD, Massachusetts General Hospital & Harvard Medical School

The GeM-HD Consortium operates with the conviction that clues to the mechanism of HD resulting in disease-modifying treatments can be gleaned from the genetic make-up of the HD population. A defining feature for understanding HD pathogenesis is the striking relationship between the HTT CAG-repeat length and age-at-onset of diagnostic signs. Together with observations from HD individuals with two expanded CAG alleles and HD heterozygotes with infrequent sequence variation at the 3’end of the repeat, genome-wide association studies (GWAS) of the HD population have led to a two-component model of HD pathogenesis in which further somatic expansion of the inherited CAG repeat in vulnerable cells triggers a toxicity mechanism. The nature of the toxicity mechanism remains uncertain. It could act at the DNA, RNA or protein level. Instability of long CAG repeats, well-documented in both humans and model systems, displays wide variation in different cell types. Similarly, individual alleles at DNA maintenance loci that influence this process, and thereby modify HD onset, can have differential impact on cognitive and motor phenotypes, arguing for cell specificity of modifier effects. This is particularly evident in results of GWAS to identify modifiers of HTT CAG-repeat instability in blood that reveal an overlapping set of modifiers with significant differences from the clinical studies. The latter have also identified several modifier loci not directly involved in DNA maintenance that are candidates for influencing toxicity rather than DNA instability. These too display evidence of cell specificity, suggesting the possibility of different toxicity mechanisms. Taken together, the HD genetic studies indicate that the vulnerability of a cell type depends on: 1) its rate of CAG-repeat expansion (a product of its inherent predisposition to expansion and the presence of particular modifier alleles); 2) the CAG length required to trigger toxicity; and 3) the toxicity mechanism(s). As with CAG-repeat instability, these later two criteria are likely to vary between cell types (as evidenced by neuronal vs. non-neuronal cells in HD individuals) and be differentially modifiable by other genes. Expanded genetic analysis of the HD population offers the promise of yet more detailed dissection of HD pathogenesis.

Top

Novel features of Huntington’s disease revealed by comprehensive transcriptional and epigenetic profiling of cell types in the human striatum and cerebral cortex

Nathaniel Heintz, PhD, The Rockefeller University

Tissue-specific somatic expansion of the mutant huntingtin (mHTT) CAG-repeat tract and regional degeneration of the brain are key features of Huntington’s disease (HD). Despite a wealth of information regarding these processes in animal models and human tissue, the relationships between somatic CAG expansion, the death of specific cell types in the human brain, and the molecular events associated with these processes have not been established. We have employed novel fluorescence-activated nuclear sorting (FANS, sFANS) and deep transcriptional and epigenetic profiling to gain insight into the properties of striatal and cortical cell types in the brains of HD and control donors. Our data demonstrate that extensive somatic expansion of mHTT CAG tract occurs in vulnerable neurons and in a subset of resilient neuron types in the striatum and cerebral cortex. We find that striatal medium spiny neurons (MSNs) are prone to somatic CAG-repeat expansion even in the absence of mHTT toxicity, and that this may be due to elevated MSH2 and MSH3 levels in MSN nuclei. We identify a single Layer 5 pyramidal cell type as selectively vulnerable in the human motor cortex in HD, and demonstrate that this vulnerable L5a pyramidal cell type projects directly to the striatum in rhesus macaques. These findings, and molecular and circuit-based mechanisms that may contribute to HD pathophysiology in humans, will be discussed.

Top

Somatic expansion of the huntingtin CAG repeat and its biological sequelae

Steven A McCarroll, PhD, Harvard Medical School & Broad Institute

Longstanding observations of HTT CAG repeat-length mosaicism in persons with Huntington’s disease (HD) – and the discovery of common genetic modifiers of HD onset in genes that encode DNA-repair proteins – have suggested a potential role for somatic expansion of the HTT CAG repeat.
To better understand HTT-CAG-length mosaicism and its relationship to HD and neuronal biology, our research team developed a molecular approach for sequencing the HTT CAG repeat in individual cells alongside genome-wide patterns of RNA expression in those same cells. We have now applied this approach to analyze tens of thousands of individual neurons from brain tissue of persons with HD.
We find that somatic expansion of the HTT CAG repeat is highly cell-type-specific and is most extensive in exactly the same cell types that later degenerate in HD. Striatal spiny projection neurons (SPNs or MSNs) and cortical projection neurons exhibit large somatic expansions, while other types of cortical and striatal neurons and glia exhibit no or much-more-modest expansion. Detailed aspects of cell-type-specific vulnerability – such as the faster loss of D2-SPNs (iSPNs) relative to D1-SPNs (dSPNs) – are also reflected in these cell types’ relative average rates of somatic repeat expansion.
We can now also analyze “allelic series” of many neurons from the same patient’s brain, using their variation in HTT-CAG length to measure the relationship of HTT-CAG length to neuronal states. Our analyses compare individual neurons that live nearby in the same tissue, controlling for disease-related and technical factors that usually confound inter-individual comparisons. We find that HTT-CAG length associates with profound changes in neuronal states, but only after substantial somatic expansion. From these data, we believe that “mutant HTT” is benign in the form in which it is generally inherited, becoming pathogenic later in life after substantial somatic expansion.
An implication of these and other results I will discuss is that therapies that seek to interfere with huntingtin expression or its downstream sequelae may have some key challenges that were previously hard to appreciate, and that the opportunity to improve outcomes by slowing or stopping somatic expansion may be significantly greater than we had realized.

Top

VO659, an allele-preferential CAG repeat-targeting ASO with therapeutic potential for individuals living with polyglutamine disease

Nicole Datson, PhD, VICO Therapeutics

Huntington’s disease (HD) and spinocerebellar ataxias (SCAs) belong to a group of polyglutamine (polyQ) diseases caused by a CAG-repeat expansion, leading to the expression of mutant proteins with a toxic gain of function. VICO Therapeutics has developed VO659, an allele-preferential CAG repeat-targeting antisense oligonucleotide (ASO) that acts via a dual mechanism of action (inhibition of translation, induction of exon-skipping). Non-clinical data obtained in disease mouse models of HD, SCA1 and SCA3 have demonstrated that VO659 reduces levels of mutant polyQ proteins and improves the disease phenotype, including improvement of motor performance in the R6/2 and Q175 mouse models of HD. In addition, VO659 was shown to reduce mutant HTT protein in patient fibroblasts with a clear CAG repeat-length dependency, with the most pronounced allelic preference observed in fibroblasts derived from juvenile HD patients.
Benchmarking experiments with a non-allele preferential competitor ASO for HD showed comparable in vitro efficacy in HD fibroblasts from adult-onset HD patients, but a better efficacy in juvenile HD fibroblasts with longer CAG repeats. Moreover, VO659 had a favorable PK profile, with higher levels throughout the mouse brain and a 3-4 fold longer brain half-life. GLP tox studies in nonhuman primates (NHPs) demonstrated a widespread CNS biodistribution and long half-life of more than 6 months in the NHP brain upon repeated intrathecal dosing. PK/PD modelling of in vivo PoC data in disease mouse models, PK data in NHPs and in vitro efficacy data, in combination with the safety profile in NHPs, supports transition to a first-in-human, phase 1/2a basket trial (EudraCT 2022-001314-19) of VO659 that has been initiated in patients with HD, SCA1 and SCA3.
VO659 is the first investigational treatment with the potential to treat all nine polyglutamine (polyQ) diseases to be tested in the clinic. We hypothesize that VO659’s allele-preferential mechanism of action enables a broader therapeutic window than non-allele preferential approaches.

Top

A new approach to HTT lowering using a C16-siRNA conjugate

William Cantley, PhD, Alnylam Pharmaceuticals

Huntington’s disease (HD) is caused by the aberrant expansion of a CAG-repeat sequence within the first exon of the huntingtin gene. While the exact mechanisms that lead to neurodegeneration and the relentless phenotypic progression of HD are not well understood, the toxicity resulting from the accumulation of the products of these expanded repeats is considered a key pathophysiological mechanism. As a result, HTT lowering has been a leading approach for HD drug development for many years. Unfortunately, a number of therapeutic HTT-lowering candidates have encountered platform limitations during clinical testing, leaving the possible benefits of HTT lowering unresolved. This issue is further compounded by the uncertainty over how much mutant HTT lowering may be necessary to provide therapeutic benefit to patients, and to what degree lowering of wtHTT can be tolerated as the upper limit of reduction remains unknown in the human CNS. Over the past 20+ years Alnylam has developed and optimized a leading platform for RNAi therapeutics, featuring durable silencing of liver-expressed genes resulting in several approved products for high unmet need indications. In 2018, we expanded our focus from liver into the CNS, with HTT lowering among our initial projects. Here we are excited to present some early data from those efforts. We discuss our CNS delivery platform and characterization of a C16-siRNA conjugate in NHP, where HTT lowering was well tolerated and sustained for at least 3 months.

Top

Advancing the science of PROTAC mutant huntingtin degraders

Adam W Hendricson, PhD, Arvinas Operations, Inc.

PROteolysis TArgeting Chimera (PROTAC) small molecules simultaneously bind an E3-ubiquitin ligase and a target protein, thereby exploiting the ubiquitin-proteasome system to selectively degrade the target via an iterative mechanism. Targeted protein degradation is an appealing option in dominantly inherited, monogenic diseases, where selectively eliminating the gene product of a toxic-gain-of function mutant allele is desired, while sparing the gene product of the unaffected allele. Huntington’s disease (HD) is a monogenic disease caused by expansion of a polyglutamine tract in the huntingtin protein resulting in an abnormal mutant protein (mHTT), which gradually damages neurons through a number of possible mechanisms and is ultimately a terminal condition with no known disease-modifying treatment. Notably, recent animal studies and human clinical trials have suggested a protective effect of the wild-type HTT in the HD brain. We have shown that orally administered PROTAC degrader molecules can cross the primate blood-brain barrier, bio-distribute to deep brain regions, and significantly degrade CNS targets. Here, we describe the discovery of potent mHTT-targeted PROTAC degraders that spare wild-type HTT, including mechanistic characterization, on-target potency and efficacy, and optimization in mHTT cellular systems, including rodent and human neurons. Molecular selectivity in HD mouse models and human HD will also be discussed. These data suggest that PROTAC molecules that harness cellular protein degradation machinery to reduce mHTT could represent future novel therapeutics for the potential treatment of HD.

Top

Antibodies from resilient individuals: Identifying a potential novel treatment for Huntington’s disease modification

Donna K Finch, PhD, Alchemab Therapeutics

At Alchemab, we are harnessing the power of the immune system to counter complex diseases. Using a combination of next-generation sequencing, serum proteomics, and computational discovery, we pinpoint antibodies associated with improved outcomes. Selected antibodies are characterized by their biological function and the targets that they bind. From analysis of multiple neurodegeneration cohorts, we have identified antibodies that bind HTT, which may have beneficial properties and bind to novel epitopes which may not be easily discovered using more traditional antibody discovery routes. We have characterized the functional activity and characteristics of our lead HTT antibody across multiple platforms, which suggests potential for a disease-modifying mechanism of action and utility as a candidate therapeutic approach for Huntington’s disease. In addition, our platform technology has the potential to deliver additional target discovery and reveal novel biology in this devastating disease.

Top

Vectorized antibodies effectively target mutant HTT protein for the treatment of Huntington’s disease

Presentation not made available by presenter

Pavlina Konstantinova, PhD, MBA VectorY B.V.

Protein misfolding is a pathogenic feature of multiple neurodegenerative diseases, including Huntington’s disease (HD). As the pathogenic mechanisms usually include a loss of function of the wild-type protein and a gain of toxicity by misfolded and aggregated proteins, therapeutic strategies are required to specifically target only the toxic protein species whilst preserving the function of the native protein.
In HD, the CAG-repeat expansion in the huntingtin (HTT) gene results in the translation of a mutant protein with an expansion of glutamines (polyQ). In neuronal cells, the mutant HTT protein aggregates form soluble oligomers and insoluble inclusions which induce toxicity and ultimately cause cell death. VectorY develops Vectorized Transformative antibodies, called VecTabs, to target and reduce the pathogenic mutant HTT protein.
We screened and identified intracellularly expressed VecTab candidates that reduce mutant soluble and aggregated HTT without interfering with wild-type HTT functions, in different in vitro models. Furthermore, we demonstrated that the VecTron technology, which is an antibody/ degron fusion protein, converts a non-degrading VecTab into a proteolytic pathway-specific degrader. The biodistribution and expression of VecTabs has been demonstrated in the central nervous system of mice and pigs without inducing any severe adverse events. In conclusion, VecTabs effectively reduce HTT aggregates in various in vitro and in vivo models, showing their promise as a potential treatment for HD.

Top

Targeting the brain with novel AAV capsid variants to reduce mutant HTT and MSH3 with siRNA-based gene therapy for Huntington’s disease

Presentation not made available by presenter

Todd A Carter, PhD, Voyager Therapeutics

Huntington’s disease (HD) is a fatal, monogenic neurodegenerative disease characterized by progressive motor, cognitive and neuropsychiatric impairment. Adeno-associated virus (AAV) gene therapy holds substantial potential for the treatment of HD, but its potential is currently limited by difficulties in delivery to the central nervous system. Disease pathogenesis is driven by a toxic gain-of-function expansion of a trinucleotide repeat in the huntingtin gene (HTT), but studies have also implicated the DNA mismatch repair protein MutS homolog 3 (MSH3). MSH3 appears to be involved in toxic somatic cell DNA expansions in the triplet repeat sequences of the mutant HTT gene, and has been reported to be a genetic modifier of disease onset and severity. Using TRACER RNA-driven directed evolution of AAV capsids, we have identified capsid variants with a greatly enhanced capacity for CNS transduction in adult nonhuman primates. Here, we demonstrate substantially enhanced delivery of a BBB-penetrant capsid in multiple species and discuss the potential to deliver siRNA-based gene therapy to reduce both mutant HTT and MSH3 broadly across the brain for the treatment of HD.

Top

The role of neurofilament light and related biofluid-based biomarkers in Huntington’s disease

Henrik Zetterberg, MD, PhD, University of Gothenburg & University College London

Genetic neurodegenerative diseases, including Huntington’s disease, provide an opportunity for targeted drug development in highly complex and clinically heterogeneous brain diseases. These neurodegenerative disorders can affect adults in their middle years, progress quickly relative to sporadic neurodegenerative diseases, and have no approved disease-modifying treatments. Huntington’s disease, caused by mutant huntingtin protein, is an active area of interventional drug trials that are testing multiple strategies to lower mutant huntingtin and/or inhibit its aggregation. These and other trials are also examining neurofilament light as a potential biomarker of disease activity and disease progression and as a therapeutic endpoint, based on the assumption that cerebrospinal fluid and blood neurofilament light levels are a surrogate for neuroaxonal damage. Reports from Huntington’s disease longitudinal studies indicate that elevated concentrations of blood neurofilament light reflect disease severity and are associated with faster brain atrophy. To better inform patient stratification and treatment response in current and upcoming clinical trials, a more nuanced interpretation of neurofilament light as a biomarker of neurodegeneration is now required, one that takes into account its relationship to other pathophysiological and topographic biomarkers of disease progression from early presymptomatic to later clinically symptomatic stages. The presentation will cover the latest news on neurofilament light and related biofluid-based biomarkers across brain diseases with the aim to improve our knowledge on how to use and interpret this class of biomarkers in clinical diagnostics and trials in Huntington’s disease.

Top

Biofluid biomarker discovery in HD – Current possibilities and limitations

Niels Henning Skotte, PhD, University of Copenhagen

The genetic nature of Huntington’s disease allows a precise diagnosis, but the use of biomarkers tracking disease progression as well as biomarkers responsive to treatment are essential to facilitate the development and evaluation of potential therapies for HD. The aims of our biomarker study were to explore several biofluids for novel protein biomarker candidates as well as substantiate previously identified protein changes. In addition, we wanted to identify potential new targets of mechanistic and therapeutic interest for further exploration. Here, we discuss our recent proteomic results and findings from human cerebrospinal fluid from healthy controls and HD gene carriers from the HDClarity cohort. We provide an overview of the most interesting protein candidates and their ability to track disease progression. Furthermore, we will discuss the current possibilities and limitations for proteomic-based biomarker discovery as well as the important next steps to validate our findings and create scientific synergy within HD research.

Top

Cholesterol and oxysterols as biomarkers for Huntington’s disease?

William J Griffiths, PhD, Swansea University

Cholesterol and its metabolism is implicated in HD. Recent studies indicate that gene therapy targeting cholesterol-related genes may offer a treatment for HD. Cholesterol is abundant in brain and because of the impermeability of the blood brain barrier (BBB) cannot be imported from, or exported to, the periphery. Essentially, all cholesterol in brain is synthesized in situ and excess cholesterol is metabolized to oxysterols, which unlike cholesterol can cross the BBB and enter the circulation. The major oxysterol exported from brain is 24S-hydroxycholesterol (24S-HC), in human this is synthesized by the neuron specific enzyme cholesterol 24S-hydroxylase. Thus, 24S-HC in plasma represents a surrogate for cholesterol metabolism in brain.
Here we have determined by liquid chromatography – tandem mass spectrometry (LC-MS/MS) the concentration of 24S-HC and other cholesterol metabolites in plasma and cerebrospinal fluid (CSF) from the HDClarity sample collection (manifest-HD n= 188, pre-manifest-HD n = 129, controls n = 83). In plasma we find that the concentration of 24S-HC is lower in manifest-HD than pre-manifest-HD or controls. In contrast, cholesterol is more abundant in manifest-HD than pre-manifest-HD. However, almost by definition, manifest-HD patients (age = 52.61 y) are older than pre-manifest-HD patients (age = 38.62), although similar to controls (age 49.08). Despite this, when both age and sex are considered as covariates 24S-HC is still significantly lower in plasma of manifest-HD patients than controls and almost significantly lower than in pre-manifest-HD patients (p=0.051).
In contrast to plasma, oxysterol content of the CSF has two sources, brain cells and from the peripheral circulation by crossing of the BBB. Therefore data from the CSF is more complex to interpret and analyses are currently ongoing.

Top

Peripheral biomarkers of somatic expansion as a therapeutic target in Huntington disease and related disorders

Darren G Monckton, PhD, University of Glasgow

Somatic expansion of the HTT CAG repeat is now established as a key step in the pathogenesis of Huntington disease (HD), and thus as a novel target for therapeutic intervention. In order to evaluate peripheral biomarkers for the in vivo efficacy of any such intervention, we have performed a detailed cross-sectional and longitudinal analysis of the dynamics of somatic expansion in the blood DNA of individuals inheriting HTT CAG expansions in the disease-associated range. Using high-throughput ultra-deep sequencing we can accurately quantify the degree of somatic expansion and have established the primary parameters governing the interaction between age at sampling and the length of the inherited HTT CAG repeat in driving somatic expansion in blood. These data have confirmed that longitudinal changes are detectable, especially in individuals inheriting larger CAG repeats. However, the magnitude of such changes over the short time frames of a likely clinical trial are relatively modest, especially in the majority of HD individuals who inherit relatively small expansions in the range of 40 to 50 CAG repeats. Nonetheless, technical improvements in the assay such as increasing the amount of input DNA, reducing the number of PCR cycles and performing multiple technical replicates can increase the sensitivity of the assay. Increasing the number of biological replicates by frequent sampling may also increase sensitivity to detect changes in the rate of somatic expansion. Other readily accessible tissue sources, such as buccal cells, may also provide opportunities. Clearly, larger inherited alleles expand more rapidly. To this end we have also started to explore the potential utility of two additional loci, CTG18.1 and ERDA1, that have a high frequency (~ 5% and 15 %, respectively) of expanded alleles (50 to 100 CAG•CTG repeats) in the general population. In addition, we are also investigating the potential utility of conducting such clinical trials in related disorders such as myotonic dystrophy type 1, for which similar mutational dynamics have been implicated in the disease process, but for which the average inherited CTG•CAG allele size is typically much larger (>> 50 repeats) and thus expands much more rapidly in blood DNA.

Top

Update on mHTT-specific PET imaging in HD

Andrew Wood, PhD and Longbin Liu, PhD, CHDI

Mutant huntingtin protein (mHTT) aggregates are a hallmark of Huntington’s disease (HD). PET ligands targeting mHTT aggregates have the potential to detect pharmacodynamic response to treatments directed at lowering mHTT in the brain as well as to study the pathology of HD progression. In addition to the current clinical candidate, CHDI-180R, CHDI has developed several 2nd generation PET ligands with further optimized characteristics targeting mHTT aggregates and these ligands are being advanced to clinical trials. This presentation will review the discovery/ development process, the features of the 2nd generation ligands, and current clinical progress.

Top

Data-driven modelling of Huntington’s disease progression:
Interpretation, staging and prediction

Peter Wijeratne, PhD, University of Sussex

Mixed modality biomarkers, such as imaging and biofluids, can provide powerful markers for tracking disease progression in neurodegenerative diseases. However, individual data can be confounded by inter-individual variability, measurement noise and individual disease stage. Disease-progression modelling uses probabilistic methods to untangle confounding effects and hence learn patterns of disease-related changes directly from data. Here we apply recent developments in disease progression modelling to i) uncover insights into Huntington’s disease progression, and ii) provide new staging and predictive utility for clinical applications. We also show a new data-driven probabilistic model, the temporal event-based model (TEBM), and demonstrate that it can provide complimentary information to the Huntington’s Disease Integrated Staging System (HD-ISS).

Top

Brain huntingtin and tominersen levels following intrathecal administration of tominersen in manifest Huntington’s disease

Blair R Leavitt, MD, CM, FRCPC, University of British Columbia

Objective: To report the results of a post-mortem brain analysis in four individuals with Huntington’s disease (HD), including an individual from the GEN-EXTEND open-label extension (OLE) study receiving tominersen intrathecally (IT). Tissue levels of huntingtin (HTT) mRNA and tominersen in multiple brain regions will be assessed.
Background: HD is caused by a genetic mutation that results in production of mutant huntingtin (mHTT) which is measurable in cerebrospinal fluid (CSF). Tominersen is an IT administered antisense oligonucleotide (ASO) that targets HTT RNA and has demonstrated dose and exposure-dependent decreases in CSF mHTT protein. The GEN-EXTEND study (NCT03842969) was open to adults who previously participated in any tominersen clinical study, and this post-mortem analysis represents the first human data on brain HTT mRNA and tominersen levels in an individual treated with IT tominersen.
Design/methods: An individual with manifest HD from the GEN-EXTEND study who was receiving 120 mg tominersen every 4 weeks elected to undergo medical assistance in dying (MAID). According to the patient’s wishes, post-mortem brain samples were collected approximately one hour following the MAID procedure. Coronal brain sections were collected and immediately frozen on dry ice. Matched brain sections from three HD individuals naïve to tominersen (controls) of similar age, sex, CAG length, and post-mortem interval were obtained from the UBC HD Biobank. Brain sections were stored at -80 degrees until punch biopsies were taken from multiple regions for analysis.
Results: Our preliminary analysis of HTT mRNA and tominersen levels from the individual participating in the OLE will be presented and compared to control subject data and to the levels predicted based on PK/PD modeling respectively.
Conclusions: These results provide the first human data on brain levels of tominersen and HTT mRNA after IT administration of tominersen. The comparison of this data to control brains and to the model predictions provides critical direct insight into the relationship between tominersen IT dosing, brain biodistribution, and pharmacodynamic effect of the ASO in an individual treated with tominersen.

Top

Hard edges for clinical trial planning in Huntington’s disease

Jeffrey D Long, PhD, University of Iowa

Clinical trial planning is challenging in Huntington’s disease (HD), especially due to a lack of a target treatment effect size. A recent efficacy success in Alzheimer’s disease (AD) can perhaps provide a touchstone for trial planning. Using data from Enroll-HD, IMAGE-HD, PREDICT-HD, and TRACK-HD, the results of an analysis are presented to explore potential limitations or “hard edges” in HD study design. Focus is on the HD-ISS Stage 2, as this stage might represent a critical treatment window in progression. Several endpoints are considered, including clinical and imaging outcomes. The evidence suggests that if the HD target treatment effect is about what was found in the AD study, then the sample size of the proposed trial will be unconformably large. The situation will be exacerbated by a placebo response, delayed treatment effect, dropout, or unequal allocation to arms. The remedy for a smaller study is to have a larger effect size, but there are other mitigating factors, such as the use of a baseline prognostic score, enriching the treatment population, and using a biomarker as an outcome variable. Results are presented illustrating various study designs that incorporate these factors, in the hope of demarcating the hard edges of practical limitations.

Top

PROOF-HD pivotal phase 3 trial: Pridopidine’s outcome on function in Huntington disease

Presentation not made available by presenter

Michael R Hayden, CM, OBC, MB, ChB, PhD, FRCP(C), FRSC, Prilenia Therapeutics & University of British Columbia

Pridopidine is a well-tolerated, orally administered, small molecule selective S1R agonist in clinical development for both Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS). Human PET imaging demonstrates robust S1R occupancy by pridopidine at the clinically relevant dose (45 mg bid). In preclinical models, S1R activation by pridopidine restores mitochondrial-associated ER membrane (MAM) integrity and improves a wide variety of downstream cellular pathways impaired in HD, leading to neuroprotection.
In the exploratory PRIDE-HD Phase 2 trial, pridopidine 45 mg bid demonstrated a beneficial effect vs placebo (Δ0.87, p=0.0032) on TFC at Week 52 (pre-specified exploratory endpoint). Post-hoc analysis shows this effect is driven by mild to moderate HD subjects (TFC7-13, Δ1.16, p=0.0003). TFC maintenance was associated with annual stabilization of plasma neurofilament light chain (NfL) levels (post-hoc, Δ from baseline in log2 pg/mL NfL -0.06 vs. +0.1 in placebo).
PROOF-HD is a multicenter, global double-blind, placebo-controlled, Phase 3 trial assessing pridopidine 45 mg bid in HD patients (TFC 7-13). Primary endpoint is mean change from baseline to week 65 in TFC. Secondary endpoints include change to week 65 in composite UHDRS (cUHDRS), proportion of subjects with no TFC decline and changes in Q-Motor and Total Motor Score. Change in plasma NfL levels is an exploratory endpoint.
PROOF-HD completed enrollment of 499 patients ahead of schedule in October 2021. As of February 14th, 2023, low dropout (40/499, 8.0%) is consistent with pridopidine’s favorable tolerability and safety profile. A total of 70 SAEs have been reported and none were deemed related to the study drug. In February 2023, an independent safety monitoring committee (SMC) reviewed all unblinded safety data and concluded that no safety signals of concern emerged. Top line results are expected April 2023 and will be presented.

Top

Preliminary data from an interim analysis of the VIBRANT-HD Ph2b study and rationale for termination of development of branaplam

Beth Borowsky, PhD, Novartis Pharmaceuticals

Branaplam is an orally available small molecule mRNA splicing modulator that has been explored in both children with SMA and adults with HD. VIBRANT-HD is a Ph2b study to investigate the safety, tolerability, pharmacodynamics and pharmacokinetics of branaplam given as weekly oral doses in people with early manifest HD. In August of 2022, a temporary suspension in dosing was announced due to signs and symptoms of peripheral neuropathy in some study participants. In December of 2022, the decision was made to discontinue development of branaplam as a potential treatment for people with HD. This decision followed a review of unblinded follow-up data from VIBRANT-HD indicating that the benefit:risk of branaplam in HD was not positive. We will present the initial observations leading to the pause in dosing and the decision to terminate development. We will also describe the plan for continued follow-up of study participants. Finally, we will present preliminary data from an interim analysis of the VIBRANT-HD study.

Top

An update on HD-GeneTRX1 and HD-GeneTRX2: Phase I/II clinical trials of AMT-130, a gene therapy for early-stage HD

Talaha Ali, MD, uniQure

Huntington’s disease (HD) is a gradually progressing, irreversible, neurodegenerative disorder leading to disability and death, with no disease-modifying therapies. uniQure is conducting a phase I/II clinical trial in the US (HD-GeneTRX1; NCT04120493) and EU/UK (HD-GeneTRX2; EudraCT 2020-001461-36) of AMT-130 (AAV5-miHTT) with an AAV-based gene therapy designed to reduce total huntingtin protein following intrastriatal administration.
HD-GeneTRX1 is a Phase I/II randomized, double-blind, sham-control, first-in-human trial exploring the safety, tolerability, and efficacy signals of AMT-130 in adult early-stage manifest HD patients in the US, with 5 years total follow-up (12 months blinded). A total of 26 patients are randomized into low-dose cohort 1 [Treatment: (6×1012 gc/patient), n=6; Sham, n=4] and high-dose cohort 2 [Treatment: (6×1013 gc/patient), n=10, Sham, n=6)] groups in HD-GeneTRX1. Individuals with a sham surgery arm eligible for crossover at 1 year (n=10) have been randomized for treatment or sham (control). HD-GeneTRX2 is the European Phase Ib/II open-label study of AMT-130 exploring the safety, tolerability, and efficacy signals of AMT-130 in adult early-stage manifest HD patients. A total of 10 patients in Europe, HD-GeneTRX2, [cohort 1, low-dose treatment (6×1012 gc/patient), n=6; cohort 2, high-dose treatment (6×1013 gc/patient), n=4] have been randomized for treatment.
Patients in the treatment group received a single administration of AMT-130 through MRI-guided, convection-enhanced stereotactic neurosurgical delivery directly into the striatum (bilaterally in the caudate and putamen). In HD-GeneTRX1, a planned Cohort 3 will address the objective of shortening the surgical procedure time.
Observations from 12 months of follow-up on the low-dose US cohort indicate that AMT-130 was generally well-tolerated with the most common adverse events being post-procedural headache, post-lumbar puncture syndrome, procedural complication and procedural pain. Two observed serious adverse events, unrelated to AMT-130, an upper extremity deep vein thrombosis and brief post-operative delirium, were observed and both resolved. CSF NfL increased as expected following surgical administration of AMT-130 and neared baseline at 12 months. Trends in CSF mHTT lowering support target engagement at the low-dose of AMT-130.
At HDTC 2023, we will provide updates on recruitment status, Data and Safety Monitoring Board (DSMB) updates, planned Cohort 3, and overall safety.

Top

GENERATION HD2 update and further learnings from GENERATION HD1

Peter McColgan, MD, PhD, F. Hoffmann-La Roche Ltd

Tominersen is an antisense oligonucleotide that lowers huntingtin (HTT) protein. GENERATION HD2 is an ongoing Phase II dose finding study of tominersen, evaluating safety, biomarker profiles and efficacy in individuals with prodromal and early manifest HD. We will provide an update of the current status of GENERATION HD2.
The design of GENERATION HD2 was informed by GENERATION HD1, a previous Phase III study of tominersen where post-hoc analyses identified potential benefit in younger individuals with less disease burden at lower tominersen exposure. GENERATION HD1 was stopped early based on an overall benefit-risk assessment and this data has been presented previously; the wealth of data from GENERATION HD1 provides a unique and valuable resource that can be used to inform therapeutic development in HD more broadly.
GENERATION HD1 has highlighted the importance of neurofilament light protein (NfL) as an exploratory biomarker for HTT-lowering therapies. In GENERATION HD2 a new clinical trial assay (CTA), developed by Roche Diagnostics that runs on the fully automated Elecsys platform, will be used to monitor plasma NfL. We will provide details on this and compare and contrast this with current approaches.
We will also showcase how we have been using the data collected in GENERATION HD1 to inform clinical trial design in HD.

Top