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Gagnon K.T.,Southwestern Medical Center | Pendergraff H.M.,Southwestern Medical Center | Deleavey G.F.,McGill University | Swayze E.E.,Isis Pharmaceuticals | And 10 more authors.
Biochemistry | Year: 2010

Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD. © 2010 American Chemical Society.

Capaldi D.,Isis Pharmaceuticals | Ackley K.,Girindus America Inc. | Brooks D.,Regado Biosciences | Carmody J.,ZIOPHARM Oncology | And 11 more authors.
Drug Information Journal | Year: 2012

This article, which is the first in a planned series intended to address chemistry, manufacturing, and control (CMC) aspects of therapeutic oligonucleotides, examines the topic of specifications for active pharmaceutical ingredients (APIs). The authors attempt to present basic scientific considerations for the broadest range of oligonucleotide APIs. Tests and analytical methods suitable for the control of single- and double-stranded oligonucleotide APIs and conjugated oligonucleotide APIs are discussed. © The Author(s) 2012.

Alam M.R.,Girindus America Inc. | Thazhathveetil A.K.,Northwestern University | Li H.,LaserGen | Seidman M.M.,U.S. National Institute on Aging
Methods in Molecular Biology | Year: 2014

Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site. Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity. © 2014 Springer Science+Business Media, LLC.

Nikcevic I.,University of Cincinnati | Wyrzykiewicz T.K.,Girindus America Inc. | Limbach P.A.,University of Cincinnati
International Journal of Mass Spectrometry | Year: 2011

Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts of the full-length product. When compared with low resolution LC-MS, ∼60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling. © 2010 Elsevier B.V. All rights reserved.

Girindus America Inc. and Childrens Hospital Medical Center | Date: 2011-06-01

A method for preparing an enantiomeric chromane, by asymmetrically hydrogenating a chromene compound in the presence of an Ir catalyst having a chiral ligand. The method includes the enantioselective preparation of enantiomeric equol. A preferred Ir catalyst has a chiral phosphineoxazoline ligand. Enantiomeric chromanes of high stereoselective purity can be obtained.

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