Volume 16

HD Insights Volume 16 (PDF)

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2016: Advances and Challenges in HD Therapeutic Development

By: Meredith A. Achey, BM

The year 2016 brought a number of notable developments in HD research and therapeutic development. While an approved, disease-modifying treatment remains elusive, planning and execution of first-in-human trials investigating some of the most promising advances in therapeutic development continued in earnest. Improvements to existing symptomatic therapies moved another step closer to patient use. Researchers moved the boundaries of our understanding of neurodegenerative disease forward to propose new therapeutic targets and new directions for future work. There were disappointments for the HD community as well, with the failure of several promising compounds to show improvement in human trials; however, overall, 2016 brought the HD community a few steps closer to treatments that might slow or reverse the course of HD.

Several clinical trials concluded in 2016, with mixed results. The announcement of the results of First-HD brought hope for the approval of an improved drug treatment for chorea for HD patients. The FDA requested additional information before approving deutetrabenazine, and Teva resubmitted their New Drug Application (NDA) in October 2016. The company hopes to see the drug approved in the first half of 2017.1 Teva’s PRIDE study investigating a higher dose of pridopidine also took an interesting turn in 2016, as a preliminary analysis showed that the drug failed to improve Total Motor Score in HD patients, but may have been associated with improved Total Functional Capacity at 52 weeks. Teva elected to extend the trial to 52-weeks after they discovered that the drug slowed disease progression in model systems.2,3 And finally, in the last weeks of 2016, Pfizer ended the open-label extension of the Amaryllis trial after the drug showed no clinical benefit (see Brief report).4

Ongoing and new clinical trials in 2016 included some of the first gene-targeted approaches to be investigated in HD, as well as novel approaches to slowing the disease’s progression. Ionis continues its Phase I study using intrathecal infusions of antisense oligonucleotide (ASO) IONIS-HTTRx (see HD Insights, Vol. 13), and thus far the compound has been well-tolerated.5 Azevan Pharmaceuticals announced the initiation of the Phase II STAIR trial, funded through the NeuroNEXT initiative, which will evaluate the safety and tolerability of SRX246 in HD patients with irritability.5

Trials of immune-related compounds approved for, or showing promise in other CNS disorders in HD patients continued throughout 2016. Teva continues to evaluate laquinimod, an immune modulator, through the Legato-HD trial.5 The HSG and Vaccinex also plan to conclude the SIGNAL trial of monoclonal antibody VX15/2503 in 2017, as an August 2016 analysis showed no safety issues that would necessitate stopping or modifying the trial.6

Gene-targeted therapies in preclinical development came to the forefront in 2016. While IONIS-HTTRx is an ASO currently in clinical trials, WAVE Life Sciences also has a preclinical development program with which they hope to begin human trials in 2017, using targeted ASOs to selectively silence the mutated gene (see HD Insights, Vol. 15). Voyager Therapeutics, uniQure, Spark Therapeutics, and academic researchers including Dr. Beverly Davidson at the Children’s Hospital of Philadelphia, are focusing their gene-silencing efforts on viral vector-based delivery systems, using adeno-associated viruses to infect cells with a gene-silencing piece of genetic material.7-10

Looking ahead, 2017 promises to be an exciting year for the HD community. While nothing is certain and many potential therapies are still only in the early stages of human trials, the continuing exploration of novel approaches to treat the symptoms and the progression of HD will bring many new insights, and may continue to provide hope for patients and families that one day, there will be more treatments that can make a difference.

1Teva Announces FDA Acceptance of Resubmitted New Drug Application for SD-809 for Treatment of Chorea Associated with Huntington Disease [press release]. Jerusalem: Business Wire, Oct. 20, 2016. Available at http://www.businesswire.com/news/home/20161020005246/en/

2Teva Announces Results from Exploratory 52-Week Phase 2 PRIDE-HD Study of Pridopidine in Huntington Disease [press release]. Jerusalem: Business Wire, Sept. 19, 2016. Available at http://www.businesswire.com/news/home/20160919005508/en/.

3Carroll J. Sorry folks, the PRIDE-HD trial did NOT show that Pridopidine slows the progression of Huntington’s disease. HDBuzz Sept. 30, 2016; http://en.hdbuzz.net/227. Accessed Jan. 10, 2017.

4Wild E. Pfizer Amaryllis trial ends in disappointment: no improvement in Huntington’s disease symptoms. HDBuzz Dec. 16, 2016; http://en.hdbuzz.net/229. Accessed Jan. 10, 2017.

52016 Research Report. Huntington’s Disease Society of America. 2016. Available at http://hdsa.org/wp-content/uploads/2016/12/HDSA_RsrchInvstRpt2016.pdf Accessed Jan. 9, 2017.

6Vaccinex, Inc. Announces Continuation of the SIGNAL Clinical Trial [press release]. Global Newswire, Sept. 9, 2016. Available at https://globenewswire.com/news-release/2016/09/09/870828/0/en/Vaccinex-Inc-Announces-Continuation-of-the-SIGNAL-Clinical-Trial.html.

7uniQure’s technology: Excellence in gene therapy through innovative modular technology, proprietary manufacturing and the experience to achieve success. 2016; http://www.uniqure.com/gene-therapy/uniqure-technology.php. Accessed Jan. 9, 2017.

8Voyager Therapeutics. Product Pipeline. 2016; http://www.voyagertherapeutics.com/programs.php. Accessed Jan. 10, 2017.

9Spark Therapeutics. Our Scientific Platform & Programs. 2016; http://sparktx.com/scientific-platform-programs/. Accessed Jan. 10, 2017.

10Davidson Laboratory – Dominant Neurodegenerative Disease.  http://davidsonlab.research.chop.edu/research_dominent.php. Accessed Jan. 22, 2016.

Brief report: Pfizer Amaryllis trial ends: negative results lead to termination of extension study

By: Molly J. Elson, BA

Pfizer’s Phase 2 trial of PF-02545920, a PDE-10 inhibitor, failed to find the efficacy for which it hoped. In December, Pfizer announced that the therapeutic intervention in its much-anticipated Amaryllis trial produced negative results. The drug failed to show significant improvement in movement, cognition, or behavior and, as a result, Pfizer has terminated the open-label 12-month extension study. The trial included 271 individuals with HD in five countries.

Despite negative results, Amaryllis has advanced our understanding of HD. “In my view, it was an important trial to run and Pfizer did all the right things… The trial itself was efficiently run, rapidly analysed, and the results released to the community without spin or any attempt to go fishing for positive news… The whole PDE-10 programme has taught us a lot about the neurobiology of HD, and how the communication between neurons is altered in the disease… the dataset from the trial will be a huge and valuable resource to help us understand the HD brain and design even better trials in the future,” says Dr. Ed Wild, MRC Clinician Scientist at UCL Institute of Neurology and Editor-in-Chief of HDBuzz, one of the trial’s lead investigators.

Highly Cited HD Research

In this edition of HD Insights, we take a look at impactful HD research articles of 2015. We searched Thomson Reuters’ “Web of Science” service in late 2016 to identify the five most highly cited original research articles reporting on original HD research published in 2015. We invited the corresponding authors to update us on their research and received the following responses.

Selective autophagy in HD

By: Zhenyu Yue, PhD

Highly Cited Article: Lim J, Lachenmayer ML, Wu S, et al. Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates. PLoS Genet. 2015 Feb 27;11(2): e1004987.

Autophagy offers neuroprotection by the clearance of misfolded or aggregated proteins and damaged organelles; therefore, manipulating autophagy has been investigated for its potential as a therapeutic target for neurodegenerative diseases associated with protein aggregates. HD exemplifies such illness in the nervous system, as it is the result of a pathogenic polyglutamine (polyQ) expansion in the Huntingtin gene (HTT) that leads to the accumulation and aggregation of mutant huntingtin protein (mHTT) in neurons, and subsequent neurotoxicity. Enhancement of autophagy has been shown to promote the removal of mHTT and prevent neurotoxicity;1 therefore, drugs that promote autophagy pathways hold promise for the treatment of HD. However, the development of autophagy-related drugs for neurodegenerative disease has met tremendous hurdles. For example, the detailed mechanism whereby autophagy machinery selectively degrades protein aggregation is unclear. It also remains unclear exactly how and if autophagy is altered in the progression of HD. If autophagy is impaired in the diseased state, it has yet to be tested whether autophagy function is still reparable in the context of HD. Moreover, understanding autophagy in neurons has been particularly challenging, thereby hindering the effective evaluation and development of autophagy-related drugs.

Recent studies have demonstrated the selectivity of autophagy in the degradation of protein aggregates and injured organelles, which is mediated largely by the autophagy receptors, a group of proteins that recognize specific cargos or substrates and recruit them to autophagy machinery for degradation. p62/SQSTM1 is the best-characterized autophagy receptor. Studies from our group and others have found that multiple kinases, such as autophagy kinase ULK1, coordinate the phosphorylation of the p62 protein at two serine sites in the UBA domain (S405 and S409), modulating its dimerization and binding affinity to ubiquitinated cargoes, and subsequent degradation.2-4 In HD cell models and in the Q175 animal model, we find that the presence of mHTT causes a significant increase in phosphorylation of S405 and S409 in a polyQ repeat count- (cell models) and age- dependent (Q175 mice) manner. Phosphorylation of S405 and S409 also promotes the clearance of polyQ aggregates in cells upon autophagy induction. We have shown that ULK1-mediated phosphorylation of p62 does not depend on the mTOR pathway, which suggests that in addition to canonic autophagy regulation, ULK1 plays a role in autophagy cargo selection and recruitment through the receptor phosphorylation. Indeed, our study and others demonstrate that inhibition of mTOR triggers ULK1-mediated phosphorylation of ATG14 and beclin 1 that activates VPS34 lipid kinase, and increases autophagy function in canonical autophagy regulation.5,6

It is unclear why ULK1-mediated p62 phosphorylation as part of the selective autophagy response occurs late in the Q175 model (perhaps when aggregates are formed). This observation also suggests that mHTT escapes autophagic degradation at a young adult age due to lack of selective autophagy response. Using GFP-LC3 autophagy reporter mice, we examined autophagic activity in Q175 HD mice and found no evidence of autophagosome accumulation despite nuclear mHTT aggregate formation and p62 phosphorylation. Interestingly, in Q175 mouse brains, ULK1-mediated ATG14 and beclin 1 phosphorylation is decreased, accompanied by reduced VPS34 lipid kinase activity,5 which is essential for autophagy initiation. The evidence thus reveals imbalanced ULK1 kinase activity towards phosphorylation of different substrates, namely increase in p62 vs decrease in Beclin1/ATG14 complex in Q175 mice, in part due to the p62 sequestration of ULK1 kinase away from the Beclin1/ATG14 complex. Therefore, chronic p62 sequestration of ULK1 kinase caused by mHTT could be harmful to the mTOR signaling required for the activation of autophagy. This idea may explain the observation that p62 deletion partially alleviates pathological phenotypes in HD animal models.7

In summary, our data just begins to provide insights into the regulation of selective autophagy by ULK1 and p62 in HD or other proteotoxic stress conditions. We postulate that monitoring key autophagy protein phosphorylation and VPS34 activity could provide a novel way to assess in vivo autophagic activity and signaling regulation in the CNS, and under neurodegenerative conditions. Our study suggests the potential of selective autophagy signaling as novel drug targets for the treatment of HD.

1Yamamoto A, Yue Z. Autophagy and its normal and pathogenic states in the brain. Annu Rev Neurosci. 2014;37:55-78.

2Lim J, Lachenmayer ML, Wu S, et al. Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates. PLoS Genet. 2015 Feb 27;11(2):e1004987.

3Matsumoto G, Wada K, Okuno M, Kurosawa M, Nukina N. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol Cell. 2011 Oct 21;44(2):279-289.

4Pilli M, Arko-Mensah J, Ponpuak M, et al. TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity. 2012 Aug 2437(2):223-234.

5Wold MS, Lim J, Lachance V, et al. ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington’s disease models. Mol Neurodegen. 2016 Dec 9;11(1):76.

6Park JM, Jung CH, Seo M, et al. The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14. Autophagy. 2016;12(3):547-564.

7Kurosawa M, Matsumoto G, Kino Y, et al. Depletion of p62 reduces nuclear inclusions and paradoxically ameliorates disease phenotypes in Huntington’s model mice. Hum Mol Genet. 2015 Feb 14;24(4):1092-1105.

Spread of Htt aggregates via phagocytosis

By: Margaret M. P. Pearce, PhD

Highly Cited Article: Pearce MMP, Spartz EJ, Hong W, Luo L, Kopito RR. Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain. Nat comm. 2015 Apr 13;6:6768.

Recent research has expanded our understanding of the essential roles played by glial cells in the development and proper functioning of the nervous system, far beyond their earlier description as cellular “glue.” Glia influence neuronal excitability, regulate neurotransmitter levels, prune excess neuronal processes during development, and clear dying neurons and toxic material from the brain. In neurodegenerative diseases, activation and recruitment of glia to regions of degeneration can be both beneficial and detrimental to neuronal survival; thus, a better understanding of glial signaling pathways that become activated in the disease state could aid the development of more successful therapies for neurodegenerative diseases.

Until recently, little was known about how glia respond to the presence of pathogenic protein aggregates in diseased brains. In this study, using the tractable model organism Drosophila, we examined whether phagocytic glia regulate the load of mutant Htt (mHtt) aggregates formed in neurons in vivo. We found that steady state numbers of mHtt aggregates in olfactory receptor neurons (ORNs) are reduced by Draper,1 a scavenger receptor expressed on the surface of all phagocytes in flies. Previous studies had identified an essential role for glial Draper in clearing degenerating axons during normal development2 or after axotomy,3 but this was the first indication that Draper-dependent phagocytosis could be linked to HD pathology.

A growing body of evidence supports prion-like spreading of protein aggregates associated with HD and other neurodegenerative diseases, whereby aggregates transfer from one cell to another, and self-replicate by seeded aggregation of natively-folded versions of the same protein. While most studies have focused on aggregate transfer between neurons, it is possible that glia also contribute to prion-like spread. For this reason, we examined whether aggregates of mHtt formed in ORNs could initiate prion-like conversion of wild-type Htt expressed in the cytoplasm of glia. Remarkably, we observed that glial wild-type Htt was converted over time into an aggregated state by mHtt aggregates formed in nearby ORN axons (Figure, part a). Since wild-type Htt only aggregates when it comes into direct physical contact with already-aggregated Htt,4,5 the appearance of punctate wild-type Htt reported entry of ORN-derived mHtt aggregate “seeds” into the glial cytoplasm.

Figure 1

Figure: Prion-like transmission of Htt aggregates from neurons to phagocytic glia in the adult Drosophila brain.

(a) Confocal images showing that wild-type HttQ25 expressed in glia (green) undergoes time-dependent conversion from soluble to aggregated (punctate) in the vicinity of mutant HttQ91 aggregates that formed in ORN axons (red). Mutant HttQ91 was expressed in DA1 ORNs, whose axons terminate in the DA1 glomerulus (DA1g, solid white line) of the antennal lobe (AL, dotted white line). Scale bars = 10μm.

(b) HttQ25 aggregate formation in the DA1 glomerulus of 7-day-old adult flies was blocked in flies homozygous for the drprΔ5 null mutation (draper -/-). Scale bars = 5μm.

(c) Model for prion-like transfer of neuronal mutant HttQ91 aggregates into the cytoplasm of glia via Draper-dependent phagocytosis. Whether ORN-derived HttQ91 aggregates escape into the glial cytoplasm during phagocytic engulfment or at a later step in phagocytosis (dotted lines) is not known.

(a and b) reproduced with permission from ref. [1].

These findings indicate that mHtt aggregates undergo prion-like transfer between the cytoplasm of neurons and glia in the intact Drosophila brain. We next took advantage of the extensive genetic toolset available in Drosophila to elucidate the mechanism responsible for neuron-to-glia Htt aggregate transfer. To our surprise, induced aggregation of glial wild-type Htt was eliminated in mutant flies that lack Draper expression (Figure, part b). In addition, RNAi-mediated knockdown of several glial genes with previously identified roles in Draper-dependent phagocytosis inhibited formation of wild-type Htt aggregates in glia. These exciting findings suggest that neuronal mHtt aggregates engulfed by Draper-dependent phagocytosis can escape from the phagolysosomal system and gain access to the glial cytoplasm (Figure, part c).

Our study suggests a double-edged role for phagocytic glia in HD pathogenesis — eliminating toxic mutant Htt aggregates from neurons, but also contributing to aggregate spread between cells. Numerous additional studies in the past few years have led to increased awareness of phagocytic glia as active players in neurodegeneration, and therefore potential therapeutic targets. In mice, the Draper homolog MEGF10 regulates turnover of synapses by phagocytic astrocytes in the adult brain,6 suggesting that protein aggregate clearance from axons might occur by a similar mechanism in mammals.  Future studies into how prion-like aggregates spread between neurons and glia, and the specific neuroprotective and neurodestructive functions of phagocytic glia will help to guide new strategies in the battle against HD and other neurodegenerative diseases.

1Pearce MMP, Spartz EJ, Hong W, Luo L, Kopito RR. Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain. Nat comm. 2015;6:6768.

2Hoopfer ED, McLaughlin T, Watts RJ, Schuldiner O, O’Leary DD, Luo L. Wlds protection distinguishes axon degeneration following injury from naturally occurring developmental pruning. Neuron. Jun 15 2006;50(6):883-895.

3MacDonald JM, Beach MG, Porpiglia E, Sheehan AE, Watts RJ, Freeman MR. The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron. Jun 15 2006;50(6):869-881.

4Chen S, Berthelier V, Yang W, Wetzel R. Polyglutamine aggregation behavior in vitro supports a recruitment mechanism of cytotoxicity. J Mol Biol. Aug 3 2001;311(1):173-182.

5Ren PH, Lauckner JE, Kachirskaia I, Heuser JE, Melki R, Kopito RR. Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates. Nat Cell Biol. Feb 2009;11(2):219-225.

6Chung W-S, Clarke LE, Wang GX, et al. Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways. Nature. 11/24 2013;504(7480):394-400.

Research Round-Up

By: Lise Munsie, PhD

In pharma…

The well-tolerated, FDA-approved drug metformin is an antidiabetic drug that mimics caloric restriction. Jin and colleagues tested their hypothesis that it may also counteract pathways affected by the presence of mHTT using the striatal Q111 cell model.1 Under serum starvation conditions, the drug corrected phenotypes such as decreased ATP production, increased LDH release, and decreased mitochondrial membrane potential, and modulated the balance between mitochondrial fusion and fission genes by activating the AMPK pathway. The group confirmed that metformin crossed the blood-brain barrier, and that increased levels of p-AMPK could be found in the brain post-administration, suggesting that this drug could be used as a neuroprotectant in HD.

The drug topotecan, a topoisomerase 1 inhibitor, leads to the activation of Ube3a, a ubiquitin ligase implicated in mHTT clearance. In a recent article in Human Molecular Genetics, Shekhar and colleagues report on their study in which topetecan delivery was optimized and tested for symptomatic improvement in the R6/2 HD mouse model.2 The drug partially rescued striatal atrophy and decreased mHTT aggregate load. The group saw upregulation of Ube3a in the brain, and a decrease in wildtype and mutant htt levels. Although side effects on other affected genes must be carefully monitored, this drug may move forward as one that delays HD progression.

The Tingle group investigated the hypothesis that dysfunctional cholinergic neurotransmission through a loss of agonist contributes significantly to HD pathology.3 The group attempted pharmacological replacement using the nicotine analogue and partial agonist varenicline. Chronic exogenous administration to YAC128 HD model mice resulted in motor and behavioral improvements combined with improved neuorophysiological phenotypes. Varenicline is approved for other disorders, and its multi-targeted and direct effects makes it a promising candidate for further study.

1Jin J, Gu H, Anders NM, et al. Metformin protects cells from mutant huntingtin toxicity through activation of AMPK and modulation of mitochondrial dynamics. Neuromolecular Med. 2016 Dec;18(4):581-592.

2Shekhar S, Vatsa N, Kumar V, et al. Topoisomerase 1 inhibitor topotecan delays the disease progression in a mouse model of Huntington’s disease. Hum Mol Genet. 2016 Dec 22.

3McGregor AL, D’Souza G, Kim D, Tingle MD. Varenicline improves motor and cognitive deficits and decreases depressive-like behaviour in late-stage YAC128 mice. Neuropharmacology. 2016 Dec 23;116:233-246.

In patient studies…

Detailed genetic analysis of HD is important for improving targeted therapies. The Hayden lab reports on HD haplotypes in the Latin American population.1 By genotyping Peruvian HD patients, the group identifies the HD mutation on the A1 HTT haplotype, a haplotype also common in European HD patients. The group defines an Amerindian-specific A1 variant based on single nucleotide polymorphism (SNP) analysis in this population. This is the most frequent HD haplotype in the studied population. This finding shows a distinct genetic origin between HD patients on Amerindian and European origin in Latin America, but still a common genetic target.

Studying genes that affect the age of onset of disease can uncover pathways involved in HD progression. In a recent article, a Spanish group probes the association between SNPs in the melanocortin 1 receptor gene (MCR1) and age of onset in the Spanish HD population.2 MCR1 is involved in anti-oxidant and cell stress pathways. Variants in this allele occur at a normal frequency in the HD population. Using a multiple linear regression model, the group show that the p.R151C polymorphism, a mutation that affects the protein’s function, may decrease the age of onset of clinical signs in Spanish HD patients, which suggests this protein and pathway may be worthy of further study.

The Milek group examined cardiovascular pathology of HD in patients.3 Expression of mHTT has negative consequences in non-neuronal tissues, and heart disease may factor into deaths in HD patients. This led the group to compare individuals with HD at different stages of disease progression, and age-matched controls. Although not significant between all groups, there was an overall increased risk for coronary heart disease in HD patients. Specific measures showed arterial dysfunction including significantly decreased distensibility of the carotid arterial wall in presymptomatic HD patients. Further and larger studies will be required to elucidate full vascular pathology in HD patients.

1Kay C, Tirado-Hurtado I, Cornejo-Olivas M, et al. The targetable A1 Huntington disease haplotype has distinct Amerindian and European origins in Latin America. Eur J Hum Genet. 2016 Dec 21.

2Tell-Marti G, Puig-Butille JA, Gimenez-Xavier P, et al. The p. R151C polymorphism in MC1R gene modifies the age of onset in Spanish Huntington’s Disease patients. Mol Neurobiol. 2016 Dec 6:1-5.

3Kobal J, Cankar K, Pretnar J, et al. Functional impairment of precerebral arteries in Huntington disease. J Neurol Sci. 2017 Jan 15;372:363-368.

In pathways…

An exciting study from the Truant lab examines endogenous HTT function in live human cells.1 They identify HTT at sites of DNA damage, localized to DNA repair proteins during induced oxidative stress. This localization is dependent on the activity of ataxia telangiectasia mutated (ATM) protein, a kinase that has been shown to contribute to HD progression in mouse models. The group hypothesize that inhibiting ATM activity, which slows or inhibits mHTT recruitment to sites of DNA damage, may have therapeutically beneficial effects in HD.

Transcriptional dysregulation is a hallmark of HD. The Jones group focuses their pathway study on differential gene expression upon epidermal growth factor (EGF) stimulation in two cellular models of HD.2 The group found that regulatory genes within the TGFβ signaling pathway were differentially expressed in cells harboring the expanded allele, and many were associated with SMAD transcription factors (TFs). The group shows that stimulation with TGFβ1 activates SMAD TFs, leading to nuclear localization. This nuclear translocation is altered in cells that express mHTT. They additionally demonstrate direct binding of SMAD3 to HTT in the cells, showing that these proteins may directly affect HTT expression. The group concludes that TGFβ signaling is a possible target for disease modification and could prove to be a useful biomarker for disease progression.

HD patients have decreased levels of striatal phosphodiesterase 10 (PDE10), an enzyme that hydrolyzes cyclic AMP and GMP. Acute inhibition of PDE10 can partially restore the basal ganglia circuitry in HD models. A manuscript in Neuron3 extensively examines the neurophysiology associated with acute PDE10 inhibition in two mouse models that recapitulate the pre-existing PDE10 deficit. Using electrophysiological and proteomic approaches, the group defines how corticostriatal transmission is altered and subsequently improved in these models under PDE10 inhibition, specifically probing the striatal indirect pathway.

1Maiuri T, Mocle AJ, Hung CL, et al. Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex. Hum Mol Genet. 2016 Dec 25.

2Bowles KR, Stone T, Holmans P, Allen ND, Dunnett SB, Jones L. SMAD transcription factors are altered in cell models of HD and regulate HTT expression. Cell Signal. 2016 Dec 14;31:1-14.

3Beaumont V, Zhong S, Lin H, et al. Phosphodiesterase 10A inhibition improves cortico-basal ganglia function in Huntington’s disease models. Neuron. 2016 Dec 21;92(6):1220-1237.



NAME: uniQure


SHARE PRICE AS OF 2/17/17: $6.31

52-WEEEK RANGE AS OF 2/17/17: $5.25-$16.40



Dr. Pavlina Konstantinova, a research scientist at uniQure, spoke with HD Insights about the company’s program to develop a gene therapy treatment for HD, using micro RNAs expressed from a DNA cassette delivered via an adeno-associated viral vector. The following is an edited transcript of the conversation.

HD INSIGHTS: Tell us about uniQure.

KONSTANTINOVA: uniQure is in a unique position because we have established a scalable manufacturing platform for adeno-associated virus (AAV) vector delivered gene therapies for commercial use. We started this a number of years ago when we were developing our first product, Glybera® (alipogene tiparvovec), which was developed in the mammalian system that a lot of other researchers are using.

However, during the Glybera® development period, we recognized that the process that we were using was not scalable and was also not really stable for commercial use. So we switched from a mammalian production system to an insect-based production system a number of years ago. At the moment, we think we are in a unique position, and we are the first company that has invested in developing this large-scale manufacturing platform for AAV vector delivered gene therapies that have commercial market potential.

Glybera® has been approved by the European drug regulatory agencies for treatment of familial lipoprotein lipase deficiencies. It is not available on the American market at the moment.

HD INSIGHTS: What are uniQure’s plans for HD therapeutics?

KONSTANTINOVA: uniQure started the HD program about six years ago. We combined our expert experience with AAV production and delivery with the micro RNA-based silencing approach. Prior to HD, we worked successfully to prove the concept of our gene silencing technology for liver disease, and we published a number of articles in peer-reviewed journals. After that initial work, HD seemed to be a good candidate for the gene silencing approach.

HD is a monogenetic disease with a very well-defined nature of the primary gene mutation that results in production of mutant huntingtin. But besides that, I think HD is a devastating disease, and no treatment is currently available. We saw an opportunity to try to develop a disease-modifying therapy that can eventually be a game-changer in management of HD. Reducing mutant huntingtin expression seems to us to be the most logical step to take because doing so can be expected to significantly contribute to slowing down disease progression.

At the moment, we are developing an RNA interferencebased approach. We have engineered small molecule RNAs known as micro RNAs (miRNAs) that target human huntingtin messenger RNA, expressed from a DNA cassette incorporated into AAV vector particles. This expression cassette will be delivered to the patient, with the aim of reducing the expression of the mutant gene that results in HD.

HD INSIGHTS: Other companies are also investigating gene silencing therapies for HD. Can you tell us how your approach is different or similar to theirs?

KONSTANTINOVA: First of all, companies that are silencing the mutant gene that causes HD are trying to lower the amount of mutant huntingtin. What distinguishes us from other companies is that we are proposing to use a single-time gene therapy, while the other approaches would require multiple administrations. Also, with the gene therapy approach using AAV, we can select the vector serotype to refine the targeting and safety of the delivery system, and we have been working on refining the administration procedure in order to have a very safe and efficacious profile. I think that is really our strength the combination of the mechanism of action, the vector serotype, production platform and the administration procedure that we have selected.

HD INSIGHTS: How and where would the therapy be administered?

KONSTANTINOVA: At the moment, we envision targeting the striatal structure in the brain. This would involve intraparenchymal injection using the convection-enhanced diffusion delivery method.

HD INSIGHTS: How is your preclinical work going? Have you applied this therapy to animal models, for example?

KONSTANTINOVA: We collaborate with others in our preclinical work. We have tested the therapy in a wide range of HD animal models. A number of years ago, we started a collaboration with Dr. Nicole Déglon from CHUV Lausanne, Switzerland, who has developed an HD rat model. The first proof-of-concept experiment was performed in this model, which typically displays very acute pathology. Neurodegeneration occurs very quickly, with very severe mutant huntingtin aggregate formation.

After these experiments, we moved to the humanized mouse model, working with Dr. Amber Southwell from University of British Columbia in Dr. Michael Hayden’s lab, and we performed a number of dose escalation studies in this model. The model is unique because it has two copies of the human huntingtin gene, one mutated, one wild-type, and no murine background huntingtin.

That makes it very suitable for a gene therapy approach like ours, where the huntingtin gene would be lowered, as well as looking at the tolerability of total huntingtin lowering. We also collaborated with Charles River Laboratories, where we are testing the functional improvement in the more standardized HD rodent models.

In the last two years, most of our experiments have been in large animals. We started our HD mini-pig experiments in collaboration with Dr. Jan Motlik from IAPG, Libechov, Czech Republic. This model was developed together with CHDI. In these experiments, we deliver our gene silencing therapy in a similar manner to how we envision delivery in our clinical program. We have obtained the first data from this HD mini-pig model, showing substantial total huntingtin knock-down, as well as very good vector distribution and miRNA expression. I think that so far, we have seen an adequate safety profile. And finally, we have performed tests in healthy non-human primates to refine the delivery procedure. We have injected different brain regions with increasing vector doses and looked at vector distribution, inflammation and neurodegeneration. Based on the outcome of those experiments, we are defining our therapeutic dose in patients aiming at approximately 50% huntingtin knock-down and no associated adverse events. As you can see, we have used several animal models of HD as well as healthy non-human primates to propel our preclinical program toward the development stage.

HD INSIGHTS: Can you tell us about the clinical effect you saw from your trials in the pig model?

KONSTANTINOVA: At the moment the pig model does not show clinical symptoms of HD, so the only parameters we could assess were mutant huntingtin lowering in the CNS, as well as vector distribution and safety. We looked at huntingtin lowering in the CSF at three months and saw a trend of reduction in the treated animals. In the blood, we looked mainly at markers for immune system activation, because this is a very important safety readout for our therapy. We found transient increase in some cytokines and interleukins in the treatment groups.  The study has not yet been published, but we are hoping to submit it for publication before the CHDI meeting.

HD INSIGHTS: Do you think it is feasible in humans to be able to look at huntingtin levels or to look for huntingtin-lowering effects in either the blood or CSF?

KONSTANTINOVA: I think if it were at all possible to have a positive readout for mutant huntingtin, it would be in the CSF. Because our therapy would be administered directly into the CNS, we do not expect leakage of the gene-silencing agent into the periphery, so all the changes in mutant huntingtin that we expect would be in the CSF. I am not aware of positive measurements of mutant huntingtin in the blood or in serum at the moment. We can envisage measuring mutant huntingtin levels in the CSF as it is a biomarker for therapeutic efficacy.

HD INSIGHTS: Researchers take both allele-specific and non-allele-specific approaches to gene silencing in HD. What is uniQure currently working on and why?

KONSTANTINOVA: We published a paper in Molecular Therapy – Nucleic Acids about our evaluation of both approaches, allele-specific and total silencing;1 however, our program development within the company has focused on total huntingtin silencing. Of course, we are working on the allele-specific approach as well, but the first therapy that we would like to bring to patients would be based on total huntingtin silencing.

HD INSIGHTS: When do you plan to start testing your therapy in humans?

KONSTANTINOVA: The program is progressing very well and we are working toward a company goal to begin a clinical trial in 2018.  At the moment, we are initiating our IND-enabling studies, and we are defining our regulatory pathway. To support our clinical program, we are creating a clinical network of key opinion-leaders in the HD field, but also neurosurgeons, experts in gene therapy delivery to the brain, and brain anatomy specialists who can help us and advise us on the development of our program.

HD INSIGHTS: Do you have early thoughts on the population of individuals affected by HD that you would see as most appropriate for initial investigations?

KONSTANTINOVA: We have talked about it a lot with our key opinion-leaders. As is well-known, brain atrophy in HD patients starts years before the onset of symptoms, and neuronal degeneration precedes decline in neuronal function. If we start from this standpoint, we think that to have a high likelihood of therapeutic efficacy, we would need to treat patients as early as possible, either at the time of diagnosis or shortly after the start of manifestation of symptoms. So I think for the first trial we would aim to go as early as possible in the course of the disease. To achieve maximum therapeutic benefit, we would need to go to patients at the time of genetic diagnosis.

HD INSIGHTS: Are there other things that you would like the community to know about your approach and your gene silencing therapies?

KONSTANTINOVA: Yes, I think we already have very promising results. Combined with the production platform that we have developed, I think we can make HD gene therapy a reality in the near future. So, I think that gene silencing as a disease-modifying treatment can really be a game-changer for HD patients.

HD INSIGHTS: Thank you and your colleagues at uniQure very much for all your efforts seeking and hopefully finding treatments that will make a huge difference for HD.

1Miniarikova J, Zanella I, Huseinovic A, et al. Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington’s Disease. Mol Ther Nucleic acids. 2016;5(3):e297.

MEET THE SCIENTIST: Pavlina Konstantinova, MSc, PhD


NAME: Pavlina Konstantinova, MSc, PhD

TITLE: Director of Emerging Technologies and Huntington Disease Program Leader, uniQure

EDUCATION: MSc, Biochemistry and Microbiology, Sofia University, Bulgaria; PhD, Bulgaria Academy of Sciences, Sofia, Bulgaria; Post-doctoral research, RNAi-based gene therapy for HIV-1, Academic Medical Center, Amsterdam Netherlands; Post-doctoral research, Viral miRNA function, Duke University, Durham, NC

HOBBIES: Yoga and spending time with her family

Dr. Pavlina Konstantinova is a scientist whose research interests have centered on virology and RNA interference. She joined uniQure eight years ago to bring together these interests and develop gene therapy and delivery mechanisms. She initiated the company’s HD program several years later.


By: Meredith A. Achey, BM


COMPOUND: Artificial micro RNA targeting human huntingtin, carried by an adeno-associated virus 5 (AAV5) vector (AAV5-miHTT)

MECHANISM OF ACTION: Micro RNAs (miRNAs) target messenger RNA (mRNA) transcripts and lead to silencing of the gene by suppressing translation. uniQure’s design also incorporates DNA promoters to enhance transcription of the miRNAs in target tissues, and the AAV5 vector is especially effective in targeting liver and neuronal tissues.1 AMT-130 was selected in preclinical studies both for efficacy in lowering mutant huntingtin production and for minimizing off-target effects.2

Figure: HTT silencing gene therapy

(1) Adeno-associated viral vector silencing human HTT gene (AAV5-miHTT) binds to neuron cell-surface receptors and is internalized. (2) The viral vector is transported into the nucleus and then degrades, uncoating the miHTT transgene. (3) The miHTT transgene is expressed and processed by the endogenous RNA interference machinery. (4) The hairpin structured precursor of miHTT is transported to the cytoplasm and further processed to the final mature miHTT. (5) The mature miHTT binds HTT messenger RNA, and the RNA duplex is recognized by RNA-induced silencing complexes. (6) HTT messenger RNA is cleaved, resulting in lowering of huntingtin protein expression. Image and caption courtesy of uniQure.

1 Maczuga P, Lubelski J, van Logtenstein R, et al. Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy. Mol Ther. 2013;21(1):217-227.

2 Miniarikova J, Zanella I, Huseinovic A, et al. Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington’s Disease. Mol Ther Nucleic acids. 2016;5(3):e297.

Highlights from the Journal of HD

Assistive technology for cognition in HD

By: Marleen R. van Walsem, MSc, Emilie I. Howe, MSc and Jan C. Frich, MD, PhD

Original Article: van Walsem MR, Howe EI, Frich JC, Andelic N. Assistive Technology for Cognition and Health-related Quality of Life in Huntington’s Disease. J Huntingtons Dis. 2016 Oct 1;5(3):261-270.

Assistive technology for cognition (ATC) can be defined as any external device aimed at improving or maintaining functional abilities of individuals with cognitive impairment.1 ATC can specifically address disabilities in memory, executive functions (planning, organization and attention), and reduced psychomotor speed. ATC comprises devices that are easily accessible and readily employed by anyone (i.e. smartphones, post-it notes), and also complex high-tech devices or programs specifically developed for supporting cognitive abilities. ATCs are used as a supportive service, and have shown themselves to be a promising strategy in conditions characterized by cognitive disabilities, for example traumatic brain injury and Alzheimer’s disease.2

Progressive development of cognitive disability is a hallmark of HD. Given the nature of cognitive impairment in HD, including reduced planning and organization skills, attention deficits and reduced psychomotor speed, there may be a benefit of ATCs for patients with HD, including devices that support structuring everyday life and memory. However, to date no research has investigated the use of ATC in HD patients.

The aim of our study was to describe aspects of ATC, including ATC use, information, needs assessment, and training, in HD patients, and to explore the association between using ATC and health-related quality of life (HRQoL). We used a cross-sectional population-based study design that included 84 HD patients who lived in south-eastern Norway, who had a clinical diagnosis of HD across the five disease stages. In addition to information regarding aspects of ATC, we collected socio-demographic and clinical data including disease-specific information. A general evaluation of cognitive impairment was also performed. We used the Unified Huntington’s Disease Rating Scale (UHDRS) Total Functional Capacity (TFC) scale as a standardized measure of functional ability. Overall HRQoL was assessed using the EQ-5D Visual Analogue Scale. In order to describe the aspects regarding ATC use and provision, we used descriptive statistical analyses, and we used multivariate regression analyses to explore the association between ATC use and HRQoL.

Results of the descriptive analyses on ATC are presented in Table 1. We found that approximately one-third (36.9%) of the patients with HD used ATC. We also found that patients who used an ATC device were predominantly in stages I−III, with mild to moderate cognitive impairment. Regarding other aspects of ATC, less than half (44%) of patients had received information about ATC, less than one-third (32.1%) had undergone a needs assessment, and only one-fifth had received ATC training. Most of these patients were in stage III. Furthermore, TFC was identified to be the only variable that bore significant impact on HRQoL (β-value = -0.564; β 95% CI 1.47− 5.34; r2 = 0.142; p = 0.001) in our multivariate regression model, which explained 30% of the variance.

Our findings indicate relatively infrequent ATC use, information provision, needs assessment and training, especially considering that the progressive development of cognitive disabilities for which specialized ATC devices exist is an inevitable symptom of HD. The lack of association between ATC use and HRQoL may reflect a lack of awareness and knowledge about ATC availability and provision among healthcare professionals. Further research into the potential benefits of ATCs in supporting cognitive disabilities and thereby positively affecting functional ability and HRQoL is warranted. Future studies should employ disease-specific measures of HRQoL that may be more sensitive to disease-specific aspects of HRQoL. ATC may prove to be a beneficial addition to the existing healthcare services for patients with HD.

  Complete sample (N = 84) Stage I

(n = 12)

Stage II

(n = 22)

Stage III

(n = 19)

Stage IV

(n = 14)

Stage V

(n = 17)

Variables   n (%) n (%) n (%) n (%) n (%) n (%) P-value (2-sided)
ATC formal/ informal Formal



12 (14.3)

19 (22.6)

53 (63.1)

0 (0)

9 (75)

3 (25)

4 (18.2)

10 (45.5)

8 (36.4)

7 (36.8)

0 (0)

12 (63.2)

1 (7.1)

0 (0)

13 (92.9)

0 (0)

0 (0)

17 (100)

P < 0.0001
ATC use Yes


Used previously

30 (35.7)

48 (57.1)

6 (7.1)

9 (25)

3 (75)

0 (0)

14 (63.6)

8 (36.4)

0 (0)

7 (36.8)

12 (63.2)

0 (0)

0 (0)

11 (78.6)

3 (21.4)

0 (0)

14 (82.3)

3 (17.7)

P < 0.0001
ATC information* Yes


37 (44)

46 (54.8)

4 (33.3)

8 (66.7)

10 (45.4)

12 (54.5)

14 (73.7)

5 (26.3)

4 (28.6))

10 (71.4)

5 (29.4)

11 (64.7)

P = 0.045
ATC evaluation** Yes


27 (32.1)

56 (66.7)

0 (0)

12 (100)

7 (31.8)

15 (68.2)

12 63.2)

7 (36.8)

4 (28.6)

9 (64.3)

4 (23.5)

13 (76.4)

P = 0.006
ATC training Yes


17 (20.2)

67 (79.8)

0 (0)

12 (100)

5 (22.7)

17 (77.3)

7 (36.8)

12 (63.2)

2 (14.3)

12 (85.7)

3 (17.6)

14 (82.4)

P = 0.150

ATC: Assistive Technologies for Cognition/cognitive disabilities. Chi-squares were used to calculate overall group differences; * 1 missing in Stage V; **1 missing in Stage IV.

Table from van Walsem MR, Howe EI, Frich JC, Andelic N. Assistive Technology for Cognition and Health-related Quality of Life in Huntington’s Disease. J Huntingtons Dis. 2016 Oct 1;5(3):261-270. Published under a Creative Commons Attribution Non-Commercial (CC BY-NC 4.0) License.

1Scherer MJ, Hart T, Kirsch N, Schulthesis M. Assistive Technologies for Cognitive Disabilities. Crit Rev Phys Rehabil Med. 2005;17(3):195-215.

2Gillespie A, Best C, O’Neill B. Cognitive function and assistive technology for cognition: a systematic review. J Int Neuropsychol Soc. 2012;18(1):1-19.

Thank you to Teva Pharmaceuticals

HD Insights thanks Teva for its ongoing support

Teva CNS is committed to continued research and development of its product portfolio and to the development of medicines aimed at meeting the specific needs of the patient communities it serves. Teva’s legacy in CNS is grounded in its commitment to ongoing collaboration with academia, medical institutions and patient advocacy groups to find innovative solutions for patients who live with chronic and debilitating diseases.

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