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    Therapeutic Silencing of Mutant <em>Huntingtin</em> by Targeting Single Nucleotide Polymorphisms: A Dissertation

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    Authors
    Pfister, Edith L.
    Faculty Advisor
    Neil Aronin, MD
    Academic Program
    Neuroscience
    UMass Chan Affiliations
    Medicine
    Document Type
    Doctoral Dissertation
    Publication Date
    2012-07-06
    Keywords
    Huntington Disease
    RNA
    Small Interfering
    Polymorphism
    Single Nucleotide
    RNA Interference
    Genetic Therapy
    Congenital, Hereditary, and Neonatal Diseases and Abnormalities
    Genetic Phenomena
    Genetics and Genomics
    Mental Disorders
    Nervous System Diseases
    Neuroscience and Neurobiology
    Nucleic Acids, Nucleotides, and Nucleosides
    Therapeutics
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    Abstract
    Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder. Invariably fatal, HD is caused by expansion of the CAG repeat region in exon 1 of the Huntingtin gene which creates a toxic protein with an extended polyglutamine tract 1. Silencing mutant Huntingtin messenger RNA (mRNA) is a promising therapeutic approach 2-6. The ideal silencing strategy would reduce mutant Huntingtin while leaving the wild-type mRNA intact. Unfortunately, targeting the disease causing CAG repeat expansion is difficult and risks targeting other CAG repeat containing genes. We examined an alternative strategy, targeting single nucleotide polymorphisms (SNPs) in the Huntingtin mRNA. The feasibility of this approach hinges on the presence of a few common highly heterozygous SNPs which are amenable to SNP-specific targeting. In a population of HD patients from Europe and the United states, forty-eight percent were heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Seventy-five percent of patients are heterozygous at least one of three frequently heterozygous SNPs. Consequently, only five allele-specific siRNAs are required to treat three-quarters of the patients in the European and U.S. patient populations. We have designed and validated siRNAs targeting these SNPs. We also developed artificial microRNAs (miRNAs) targeting Huntingtin SNPs for delivery using recombinant adeno-associated viruses (rAAVs). Both U6 promoter driven and CMV promoter driven miRNAs can discriminate between matched and mismatched targets in cell culture but the U6 promoter driven miRNAs produce the mature miRNA at levels exceeding those of the vast majority of endogenous miRNAs. The U6 promoter driven miRNAs can produce a number of unwanted processing products, most likely due to a combination of overexpression and unintended export of the pri-miRNA from the nucleus. In contrast, CMV-promoter driven miRNAs produce predominantly a single species at levels comparable to endogenous miRNAs. Injection of recombinant self complementary AAV9 viruses carrying polymerase II driven Huntingtin SNP targeting miRNAs into the striatum results in expression of the mature miRNA sequence in the brain and has no significant effect on endogenous miRNAs. Matched, but not mismatched SNP-targeting miRNAs reduce inclusions in a knock-in mouse model of HD. These studies bring us closer to an allele-specific therapy for Huntington’s disease.
    DOI
    10.13028/a5gf-1e13
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/31966
    Rights
    Copyright is held by the author, with all rights reserved.
    ae974a485f413a2113503eed53cd6c53
    10.13028/a5gf-1e13
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