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      Inhibiting Expression of Mutant Huntingtin and Ataxin-3 by Targeting Expanded CAG Repeat RNAs

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          Abstract

          Many neurological disorders are caused by expanded trinucleotide repeats 1, including Machado-Joseph Disease (MJD) 2 and Huntington Disease (HD) 3. MJD and HD are caused by expanded CAG repeats within the ataxin-3 ( ATXN3) and huntingtin ( HTT) genes. Inhibiting expression of ATXN3 or HTT are promising therapeutic strategies, but indiscriminant inhibition of wild-type and mutant alleles may lead to toxicity. We hypothesized that expanded triplet repeat mRNA might be preferentially recognized by complementary oligomers. We observe selective inhibition of mutant ataxin-3 and HTT protein expression by peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers targeting CAG repeats. Duplex RNAs were less selective, suggesting an advantage for single-stranded oligomers. Inhibiting mutant HTT expression protected cultured striatal neurons from an HD mouse model against glutamate-induced toxicity. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length offer new options for treating MJD, HD, and other hereditary diseases.

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          Most cited references26

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          Design of antisense oligonucleotides stabilized by locked nucleic acids.

          The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2'-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2'-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5-4 degrees C per LNA depending on the positions of the modified residues. 2'-O-methyl nucleotides increase the T(m) by only 2'-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t(1/2) = approximately 1.5 h to t(1/2) = approximately 15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2'-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.
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            Potent and nontoxic antisense oligonucleotides containing locked nucleic acids.

            Insufficient efficacy and/or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a high-affinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA/DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA/DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.
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              A worldwide study of the Huntington's disease mutation. The sensitivity and specificity of measuring CAG repeats.

              Huntington's disease is associated with an expanded sequence of CAG repeats in a gene on chromosome 4p16.3. However, neither the sensitivity of expanded CAG repeats in affected persons of different ethnic origins nor the specificity of such repeats for Huntington's disease as compared with other neuropsychiatric disorders has been determined. We studied 1007 patients with diagnosed Huntington's disease from 565 families and 43 national and ethnic groups. In addition, the length of the CAG repeat was determined in 113 control subjects with a family history of Alzheimer's disease (44 patients), schizophrenia (39), major depression (16), senile chorea (5), benign hereditary chorea (5), neuroacanthocytosis (2), and dentatorubropallidoluysian atrophy (2). The number of CAG repeats was also assessed in 1595 control chromosomes, with the size of adjacent polymorphic CCG trinucleotide repeats taken into account. Of 1007 patients with signs and symptoms compatible with a diagnosis of Huntington's disease, 995 had an expanded CAG repeat that included from 36 to 121 repeats (median, 44) (sensitivity, 98.8 percent; 95 percent confidence interval, 97.7 to 99.4 percent). There were no significant differences among national and ethnic groups in the number of repeats. No CAG expansion was found in the 110 control subjects with other neuropsychiatric disorders (specificity, 100 percent; 95 percent confidence interval, 95.2 to 100 percent). In 1581 of the 1595 control chromosomes (99.1 percent), the number of CAG repeats ranged from 10 to 29 (median, 18). In 12 control chromosomes (0.75 percent), intermediate-sized CAG sequences with 30 to 35 repeats were found, and 2 normal chromosomes unexpectedly had expanded CAG sequences, of 39 and 37 repeats. CAG trinucleotide expansion is the molecular basis of Huntington's disease worldwide and is a highly sensitive and specific marker for inheritance of the disease mutation.
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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nature biotechnology
                1087-0156
                1546-1696
                13 April 2009
                3 May 2009
                May 2009
                1 November 2009
                : 27
                : 5
                : 478-484
                Affiliations
                [1 ]The Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Road, Dallas, TX, 75390-9041
                [2 ]The Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Road, Dallas, TX, 75390-9041
                [3 ]SIGMA-Aldrich Genopole Campus 1. 5, rue Desbruères, 91030 Evry Cedex, France
                Author notes
                [4]

                These authors contributed equally to this work

                AUTHOR CONTRIBUTIONS

                J.H. and M.M designed and performed experiments in patient-derived fibroblast cells. J.W. and J.H. designed and performed experiments in MSN cells. K.T.G. and J.C.S assisted with experiments. K.A. and S.G. supplied LNAs. D.R.C. and I.B. supervised experiments.

                [* ]Correspondence should be addressed to D.R.C. ( david.corey@ 123456utsouthwestern.edu )
                Article
                nihpa107907
                10.1038/nbt.1539
                2765218
                19412185
                235c896e-c5c2-4082-b78f-dd70ea1f52c3
                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM073042-05 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM060642-08 ||GM
                Categories
                Article

                Biotechnology
                Biotechnology

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