TricycloDNA-modified oligo-2′-deoxyribonucleotides reduce scavenger receptor B1 mRNA in hepatic and extra-hepatic tissues—a comparative study of oligonucleotide length, design and chemistry

We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in Tm, cell culture and animal experiments and compare them to their gap-matched 20-mer 2′-O-methoxyethyl (MOE) and 14-mer 2′,4′-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar Tm and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED50 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower Tm and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animal

Calibration and evaluation of AVIRIS data: Cripple Creek in October 1987

Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) data were obtained over Cripple Creek and Canon City Colorado on October 19, 1987 at local noon. Multiple ground calibration sites were measured within both areas with a field spectrometer and samples were returned to the laboratory for more detailed spectral characterization. The data were used to calibrate the AVIRIS data to ground reflectance. Once calibrated, selected spectra in the image were extracted and examined, and the signal to noise performance was computed. Images of band depth selected to be diagnostic of the presence of certain minerals and vegetation were computed. The AVIRIS data were extremely noisy, but images showing the presence of goethite, kaolinite and lodgepole pine trees agree with ground checks of the area

Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies

Competition for RISC binding predicts in vitro potency of siRNA

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2′-O-methyl and 2′-deoxy-2′-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity

Synthesis of 1-(2-aminopropyl)benzimidazoles, structurally related to the TIBO derivative R82150, with activity against human immunodeficiency virus.

A number of 1-(2-aminopropyl)-2-mercaptobenzimidazoles related to the TIBO derivatives R82150 have been prepared and tested for their activity against human immunodeficiency virus type 1 (HIV-1). These compounds were all modest inhibitors of the cytophatic effects of HIV-1 in vitro, but only very weak inhibitors of HIV-1 reverse transcriptase (RT).A number of 1-(2-aminopropyl)-2-mercaptobenzimidazole derivatives related to the TIBO derivative R82150 have been prepared and tested for their activity against human immunodeficiency virus type 1 (HIV-1). These compounds are all modest inhibitors of the cytopathic effects of HIV-1 in vitro, but were only weak inhibitors of HIV-1 reverse transcriptase (RT).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30886/1/0000555.pd

Tau-targeting antisense oligonucleotide MAPTRx in mild Alzheimer’s disease: a phase 1b, randomized, placebo-controlled trial

Tau plays a key role in Alzheimer’s disease (AD) pathophysiology, and accumulating evidence suggests that lowering tau may reduce this pathology. We sought to inhibit MAPT expression with a tau-targeting antisense oligonucleotide (MAPTRx) and reduce tau levels in patients with mild AD. A randomized, double-blind, placebo-controlled, multiple-ascending dose phase 1b trial evaluated the safety, pharmacokinetics and target engagement of MAPTRx. Four ascending dose cohorts were enrolled sequentially and randomized 3:1 to intrathecal bolus administrations of MAPTRx or placebo every 4 or 12 weeks during the 13-week treatment period, followed by a 23 week post-treatment period. The primary endpoint was safety. The secondary endpoint was MAPTRx pharmacokinetics in cerebrospinal fluid (CSF). The prespecified key exploratory outcome was CSF total-tau protein concentration. Forty-six patients enrolled in the trial, of whom 34 were randomized to MAPTRx and 12 to placebo. Adverse events were reported in 94% of MAPTRx-treated patients and 75% of placebo-treated patients; all were mild or moderate. No serious adverse events were reported in MAPTRx-treated patients. Dose-dependent reduction in the CSF total-tau concentration was observed with greater than 50% mean reduction from baseline at 24 weeks post-last dose in the 60 mg (four doses) and 115 mg (two doses) MAPTRx groups. Clinicaltrials.gov registration number: NCT03186989

Targeting Huntingtin expression in patients with Huntington's disease

Background Huntington’s disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT, resulting in a mutant huntingtin protein. IONIS-HTTRx (hereafter, HTTRx) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant huntingtin. Methods We conducted a randomized, double-blind, multiple-ascending-dose, phase 1–2a trial involving adults with early Huntington’s disease. Patients were randomly assigned in a 3:1 ratio to receive HTTRx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of huntingtin. The primary end point was safety. The secondary end point was HTTRx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant huntingtin in CSF. Results Of the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTTRx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTTRx-treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTTRx in CSF showed dose dependence up to doses of 60 mg. HTTRx treatment resulted in a dose-dependent reduction in the concentration of mutant huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and −20%, −25%, −28%, −42%, and −38% in the HTTRx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively). Conclusions Intrathecal administration of HTTRx to patients with early Huntington’s disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann–La Roche; ClinicalTrials.gov number, NCT02519036.

Enersion Tri-Generation System: Applications, Sizing, And Economics

Enersion Inc. has developed a proprietary Tri-Generation system which can provide electricity, heating, and cooling for buildings. Information about the economic and environmental viability of such a system is understandably lacking. This work provides recommendations to Enersion Inc. regarding some potential building types and locations where their Tri-Generation system may be advantageous. Publicly available software and building models, along with one author-created model, were used to generate energy supply and demand data, which were then compared to each other, to utility rates, and to utility grid greenhouse gas emissions. It was found that hospitals, vertical farms, and mid-rise apartments in California were the most reasonable fits for solar-based Tri-Generation systems while hospitals, vertical farms, and secondary schools in Ontario were the most reasonable fits for natural gas-based Tri-Generation systems. Many achieved payback in less than 10 years. These recommendations may be used by Enersion in their marketing and business strategy

The synthesis of several members of the new imidazo(4,5-d)isothiazole ring system: Novel 5:5 fused analogs of purines and purine nucleosides.

The chemistry of purines, purine nucleosides, and their derivatives has been extensively studied, and many analogs of this important class of compounds exert a significant biological effect. Within the traditional 6:5 fused framework of the purine ring system, derivatives formed by various heteroatom substitutions are common, however the 6:5 fused ring system has seldom been varied. We have investigated the substitution of the C2-N3 linkage of the purine ring system with a single sulfur atom, giving the hitherto unknown 5:5 fused imidazo (4,5-d) isothiazole ring system. Our initial investigations focused on the synthesis of several imidazo (4,5-d) isothiazoles, as well as an investigation of their chemical, physical and biological properties. A series of imidazo (4,5-d) isothiazoles have been prepared from isothiazole precursors via a strategy employing ring annulation of the appropriate isothiazole diamine. In this manner, 5-alkylthio-3-methylimidazo (4,5-d) isothiazoles were prepared from 4,5-diamino-3-methylisothiazole via an initial reaction with thiocarbonyldiimidazole, followed by alkylation with an alkyl halide. This procedure allows the synthesis of 3-unsubstituted, 3-amino, and 6-alkyl derivatives, in addition to the 6-unsubstituted and 3-methyl compounds. Ring annulation with diethoxymethyl acetate provided several 5-unsubstituted imidazo (4,5-d) isothiazoles, but was less general, and only the 3-methyl derivatives could be prepared. The chemical and physical properties of this new ring system were then investigated, and the structure of 3-methyl-5-methylsulfonyl-6-(phenylmethyl)imidazo (4,5-d) isothiazole was verified via X-ray crystallographic studies. A series of imidazo (4,5-d) isothiazole nucleosides has also been synthesized, via a coupling of the appropriate heterocycle with the requisite pentofuranosyl synthon. In this manner, 2\sp\prime-deoxyribofuranosyl, arabinofuranosyl, and ribofuranosyl imidazo (4,5-d) isothiazole nucleosides were prepared. Several of the new compounds were evaluated for their antiviral and antitumor activity in preliminary screens. None of the compounds displayed antiviral activity at non-toxic concentrations, however, several derivatives exhibited moderate cytotoxicity in both the antiviral and antitumor assays. 3-Methyl-6-($\beta$- scD-ribofuranosyl)imidazo (4,5-d) isothiazole, which is closely related to the cytotoxic nucleoside 6-methyl-9-($\beta$- scD-ribofuranosyl)purine, displayed selective antitumor activity against murine L1210 leukemic cells in vitro.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104045/1/9423329.pdfDescription of 9423329.pdf : Restricted to UM users only