Allele-Selective Thiomorpholino Antisense Oligonucleotides as a Therapeutic Approach for Fused-in-Sarcoma Amyotrophic Lateral Sclerosis.

Pathogenic variations in the fused in sarcoma (FUS) gene are associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS). As FUS-ALS is a dominant disease, a targeted, allele-selective approach to FUS knockdown is most suitable. Antisense oligonucleotides (AOs) are a promising therapeutic platform for treating such diseases. In this study, we have explored the potential for allele-selective knockdown of FUS. Gapmer-type AOs targeted to two common neutral polymorphisms in FUS were designed and evaluated in human fibroblasts. AOs had either methoxyethyl (MOE) or thiomorpholino (TMO) modifications. We found that the TMO modification improved allele selectivity and efficacy for the lead sequences when compared to the MOE counterparts. After TMO-modified gapmer knockdown of the target allele, up to 93% of FUS transcripts detected were from the non-target allele. Compared to MOE-modified AOs, the TMO-modified AOs also demonstrated reduced formation of structured nuclear inclusions and SFPQ aggregation that can be triggered by phosphorothioate-containing AOs. How overall length and gap length of the TMO-modified AOs affected allele selectivity, efficiency and off-target gene knockdown was also evaluated. We have shown that allele-selective knockdown of FUS may be a viable therapeutic strategy for treating FUS-ALS and demonstrated the benefits of the TMO modification for allele-selective applications.

Screening Splice-Switching Antisense Oligonucleotides in Pancreas-Cancer Organoids.

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated or mutated splicing factors, or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids are a promising platform to recapitulate key aspects of disease states, and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) lipofection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma organoids. This optimized delivery method allows fast and efficient screening of ASOs, e.g., those that reverse oncogenic alternative splicing. In combination with advances in chemical modifications of oligonucleotides and ASO-delivery strategies, this method has the potential to accelerate the discovery of antitumor ASO drugs that target pathological alternative splicing.

Compromised nonsense-mediated RNA decay results in truncated RNA-binding protein production upon DUX4 expression.

Nonsense-mediated RNA decay (NMD) degrades transcripts carrying premature termination codons. NMD is thought to prevent the synthesis of toxic truncated proteins. However, whether loss of NMD results in widespread production of truncated proteins is unclear. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD), features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Using a cell-based model of FSHD, we show production of truncated proteins from physiological NMD targets and find that RNA-binding proteins are enriched for aberrant truncations. The NMD isoform of one RNA-binding protein, SRSF3, is translated to produce a stable truncated protein, which is detected in FSHD patient-derived myotubes. Ectopic expression of truncated SRSF3 confers toxicity, and its downregulation is cytoprotective. Our results delineate the genome-scale impact of NMD loss. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

Direct Synthesis of Polyaryls by Consecutive Oxidative Cross-Coupling of Phenols with Arenes.

A bioinspired iron-catalyzed consecutive oxidative cross-coupling reaction between a single phenolic unit and nucleophilic arenes was developed. This sustainable transformation offers a selective synthetic strategy for the preparation of complex polyaryl compounds directly from readily available phenols. With the aid of electron paramagnetic resonance spectroscopy, it was demonstrated that the groups ortho to the phenolic functionality (whether hydrogen, methyl, or methoxy) direct the regioselectivity (ortho, para, or meta via dienone-phenol rearrangement) and chemoselectivity (C-C coupling or C-O coupling) in this multistep process.

Significant enhancement in the efficiency and selectivity of iron-catalyzed oxidative cross-coupling of phenols by fluoroalcohols.

Significant enhancement of both the rate and the chemoselectivity of iron-catalyzed oxidative coupling of phenols can be achieved in fluorinated solvents, such as 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP), 2,2,2-trifluoroethanol (TFE), and 1-phenyl-2,2,2-trifluoroethanol. The generality of this effect was examined for the cross-coupling of phenols with arenes and polycyclic aromatic hydrocarbons (PAHs) and of phenol with ß-dicarbonyl compounds. The new conditions were utilized in the synthesis of 2'''-dehydroxycalodenin B in only four synthetic steps.

Copper(I)-catalyzed intramolecular O-arylation for the synthesis of 2,3,4,9-tetrahydro-1H-xanthen-1-ones with low loads of CuCl.

As little as 0.5 mol % CuCl is sufficient to catalyze the intramolecular O-arylation of easily accessible 2-(2-bromobenzyl)cyclohexane-1,3-diones to provide the corresponding 2,3,4,9-tetrahydro-1H-xanthen-1-ones with yields ranging from 83% to 99%.

Cu(I)-catalyzed annulation for the synthesis of substituted naphthalenes using o-bromobenzaldehydes and ß-ketoesters as substrates.

Cu(I)-catalyzed reaction of o-bromobenzaldehydes with ß-ketoesters using Cs(2)CO(3) as a base and 2-picolinic acid as an additive proceeds under mild conditions and gives access to substituted naphthalenes in a single step with yields ranging from 71 to 86%. The new annulation process relies on a domino Knoevenagel condensation/C-arylation/1,2-addition/carboxylic acid cleavage. The annulation can also be achieved with o-iodobenzaldehyde.