Consensus Guidelines for the Design and In Vitro Preclinical Efficacy Testing N-of-1 Exon Skipping Antisense Oligonucleotides

Abstract

Antisense oligonucleotides (ASOs) can modulate pre-mRNA splicing. This offers therapeutic opportunities for numerous genetic diseases, often in a mutation-specific and sometimes even individual-specific manner. Developing therapeutic ASOs for as few as even a single patient has been shown feasible with the development of Milasen for an individual with Batten disease. Efforts to develop individualized ASOs for patients with different genetic diseases are ongoing globally. The N = 1 Collaborative (N1C) is an umbrella organization dedicated to supporting the nascent field of individualized medicine. N1C recently organized a workshop to discuss and advance standards for the rigorous design and testing of splice-switching ASOs. In this study, we present guidelines resulting from that meeting and the key recommendations: (1) dissemination of standardized experimental designs, (2) use of standardized reference ASOs, and (3) a commitment to data sharing and exchange.

Keywords: N-of-1; antisense oligonucleotide; exon skipping; protocol.

FIG. 1.

Ways to use ASO-mediated exon skipping to restore protein production. (A) Normally, the protein coding information is dispersed over exons in a gene. The gene transcript will contain both exons and introns. During splicing, the introns are removed, resulting in the messenger RNA (mRNA), which is translated into a protein. (B) Cryptic splicing variants cause part of an intron to be recognized as an exon and aberrantly included in the mRNA. This prevents protein production. ASOs targeting the cryptic exon can prevent inclusion into the mRNA allowing production of a normal mRNA and normal protein. (C–E) ASO-mediated skipping of constitutively spliced exons can restore the production of partially functional proteins in multiple ways: (C) by restoring the reading frame, to allow the production of an internally deleted, but partially functional protein; (D) by skipping an in-frame exon containing a nonsense or frameshifting variant, which will bypass the variant, while maintaining the reading frame, to allow the production of an internally deleted, but partially functional protein; (E) by skipping an in-frame exon containing a toxic gain of function variant, which will allow the production of an internally deleted protein that is partially functional, rather than a toxic protein. (F) For some genes, transcripts containing poison exons, short naturally occurring exons containing a stop or a frameshift, are produced. These transcripts are subjected to nonsense-mediated mRNA decay. ASO-mediated skipping of poison exons can increase the amount of functional transcripts produced and thus increase the amount of protein, which can be therapeutic for haploinsufficiency diseases. ASO, antisense oligonucleotide.