Huntington's disease (HD) is the most prevalent autosomal dominant, trinucleotide repeat neurodegenerative disease. The huntingtin gene encodes a protein of 350 kD;the disease causing mutation is an expansion of an amino-terminal polyglutamine repeat of more than 36 successive glutamines. Our broad research goal is to understand the molecular basis of HD pathogenesis to target therapy. Because HD is an inherited disease, we expected that the mutant allele will differ from wild-type by at least a single nucleotide polymorphism, thereby offering a target for gene silencing by RNAi. Thus demonstrated, our core idea is that RNA silencing is useful to selectively reduce mutant huntingtin expression and slow or block neuronal dysfunction and death in HD. We hypothesize that selective knockdown of mutant huntingtin restores normal neuronal function, but excessive silencing impairs neuronal function by interfering with essential signaling events.
Aim 1 examines in vivo in mice whether allele specific silencing can delay or prevent HD neuropathy and abnormal behaviors.
Aim 2 investigates in post-mortem HD brain post- transcriptional regulation of huntingtin mRNA, to find molecular processes that favor synthesis of mutant huntingtin mRNA. We will use a new strategy to quantify each huntingtin allelic mRNA, based on SNP heterozygosities of the alleles. Knowledge from aims 1 and 2 is essential in applying mRNA silencing to HD patients.
Aim 3 studies cellular mechanisms by which RNAi restores essential neuronal signaling activity in HD cells and the threshold for wild type huntingtin to main normal neuronal integrity.
Aim 3 combines allelic RNAi and neuronal signaling of the cysteine transporter that regulates glutathione clearance of reactive oxygen species.
Aim 4 examines the safety of administration of allele specific siRNA, by evaluating innate immunity and inflammatory responses. This proposal satisfies NINDS goals in translational science: translation of gene silencing therapeutics, early-state therapy development, and identifying mechanisms that underlie nervous system function. Innovations include targeting mRNA alleles for RNAi, use of HD mouse models that express only human huntingtin genes, quantitative measurement of huntingtin allelic mRNA based on SNP heterozygosities, deep sequencing analysis to identify 3 UTR huntingtin mRNA regulation, and zinc finger nuclease strategy to eliminate huntingtin alleles at the genomic level. This proposal applies fundamental biology to understand pathogenesis of HD and translate this knowledge into better therapies.

Public Health Relevance

Patients with Huntington's disease (HD) develop cognitive deficits, depression and movement abnormalities. Brain cells die early. We will examine how gene silencing can reduce production of the mutant huntingtin protein that causes HD, thereby preventing dysfunction and death in neurons in animal models of HD and in HD neurons in culture. We will study safety, in brain, of the molecules that initiate gene silencing. This proposal addresses treatment of HD through study of basic mechanisms of silencing the gene that causes the disease.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Cell Death in Neurodegeneration Study Section (CDIN)
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Sutherland, Margaret L
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University of Massachusetts Medical School Worcester
Internal Medicine/Medicine
Schools of Medicine
United States
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Pfister, Edith L; Chase, Kathryn O; Sun, Huaming et al. (2017) Safe and Efficient Silencing with a Pol II, but Not a Pol lII, Promoter Expressing an Artificial miRNA Targeting Human Huntingtin. Mol Ther Nucleic Acids 7:324-334
Haraszti, Reka A; Coles, Andrew; Aronin, Neil et al. (2017) Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System. Bio Protoc 7:
Nikan, Mehran; Osborn, Maire F; Coles, Andrew H et al. (2017) Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain. Bioconjug Chem 28:1758-1766
Alterman, Julia F; Coles, Andrew H; Hall, Lauren M et al. (2017) A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics. Bio Protoc 7:
Romo, Lindsay; Ashar-Patel, Ami; Pfister, Edith et al. (2017) Alterations in mRNA 3' UTR Isoform Abundance Accompany Gene Expression Changes in Human Huntington's Disease Brains. Cell Rep 20:3057-3070
Didiot, Marie-C├ęcile; Hall, Lauren M; Coles, Andrew H et al. (2016) Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing. Mol Ther 24:1836-1847
Coles, Andrew H; Osborn, Maire F; Alterman, Julia F et al. (2016) A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo. Nucleic Acid Ther 26:86-92
Liu, Wanzhao; Pfister, Edith L; Kennington, Lori A et al. (2016) Does the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon? J Huntingtons Dis 5:33-8
Choudhury, Sourav R; Harris, Anne F; Cabral, Damien J et al. (2016) Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector. Mol Ther 24:726-35
Vodicka, Petr; Mo, Shunyan; Tousley, Adelaide et al. (2015) Mass Spectrometry Analysis of Wild-Type and Knock-in Q140/Q140 Huntington's Disease Mouse Brains Reveals Changes in Glycerophospholipids Including Alterations in Phosphatidic Acid and Lyso-Phosphatidic Acid. J Huntingtons Dis 4:187-201

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