Systematic approach to developing splice modulating antisense oligonucleotides

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone

Single stranded fully Modified-Phosphorothioate oligonucleotides can induce structured nuclear inclusions, alter nuclear protein localization and disturb the transcriptome In Vitro

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects

The capacity of activated kaolins to remove colour pigments from rice bran oil: the effects of acid concentration and pre-heating prior to activation

International audienceThis study focuses on the effects of both thermal treatment (between 80 and 700 degrees C) and chemical activation (concentration of sulfuric acid between 0.3 and 2 M) of natural Ranong kaolins (ground or not) from Thailand to remove the undesirable colour of rice bran oil. The mineralogical, physical and physicochemical properties of the initial and activated kaolins are discussed in relation with the bleaching effectiveness of the activated sample investigated. Generally, the greater the temperature used before the activation step and the concentration of sulfuric acid used during activation, the greater the structural degradation of the kaolinite; Al is removed from the octahedral sheet of kaolinite and amorphous SiO2 dominates the samples. The measured maximum bleaching capacity is not necessarily obtained when using the activated kaolin having the highest specific surface area and pore volume; rather, the bleaching capacity is dependent on both alumina contents and proportion of kaolinite in the samples. Indeed, the partial preservation of the kaolinite structure is crucial to obtain a good bleaching capacity of kaolin in relation to the preservation of the aluminol sites which are likely to be involved in the adsorption of unsaturated molecules present in the rice bran oil. Moreover, as previously demonstrated, a partial leaching of Al from octahedral sheets of kaolin is also an important factor in order to obtain good bleaching capacities. Finally, the optimal preheating temperature and concentration of sulfuric acid which permit the best bleaching capacity of kaolin are reported

Improved outcome with intensive chemotherapy in paediatric acute myeloid leukaemia.

Annals of the Academy of Medicine, Singapore335 Supp

Prevalence and risk factors of retinal vein occlusion in an Asian population

British Journal of Ophthalmology92101316-131

Large-Scale Whole-Genome Sequencing of Three Diverse Asian Populations in Singapore

10.1016/j.cell.2019.09.019Cell1793736-74