Antisense Oligonucleotide Targeting of 3’-UTR of mRNA for Expression Knockdown

Abstract

With the recent conditional approval of an antisense oligonucleotide (AON) that restores the reading frame of DMD transcript in a subset of Duchenne muscular dystrophy patients, it has been established that AONs sharing similar chemistry have clear clinical potential. Genetic diseases, such as facioscapulohumeral dystrophy (FSHD), can be the result of gain-of-function mutations. Since mRNA processing in terms of termination of transcription, its transport from the nucleus to the cytoplasm, its stability and translation efficiency are dependent on key 3'UTR elements, it follows that targeting these elements with AONs have the potential to induce gene silencing. Aberrant expression of the Double homeobox 4 (DUX4) transcription factor and the downstream consequences of such expression is the hallmark of FSHD. Here we describe the bioinformatic strategies behind the design of AONs targeting polyadenylation signals and the methodologies relevant to their in vitro screening for efficacy and safety, including analysis of expression at the transcript and protein level of the specific target and downstream genes, and measurement of the effect on the fusion index of myotubes. The targeting of permissive DUX4 and MSTN are used as examples. MSTN encodes for myostatin, a negative regulator of myogenesis; the downregulation of MSTN expression has the potential to address the muscular atrophy associated with muscular dystrophies, sarcopenia, cancer and acquired immunodeficiency syndrome.

SF3B1 inhibition disrupts malignancy and prolongs survival in glioblastoma patients through BCL2L1 splicing and mTOR/ß-catenin pathways imbalances

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Mutation-Directed Therapeutics for Neurofibromatosis Type I

“…One is to target critical regulatory regions such as the translational start codon of the mature mRNA [73][74][75][76] or the polyadenylation signal in the 3 0 UTR (untranslated region). 77,78 An alternative method for inhibition of gene expression would be to design AOs to skip exons that result in an out-of-frame transcript. 79,80 Such AOs mask exonic splice enhancer (ESE) motifs in out-of-frame exons within the pre-mRNA.…”

Delivery of antisense oligonucleotide using polyethylenimine-based lipid nanoparticle modified with cell penetrating peptide

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