agileASO

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  • Designing Principles for Individualized ASOs

    Antisense oligonucleotides are typically 15-20 bases in length. There are many factors to consider when designing ASOs. Sequences too long are not easily absorbed by cells, while too short would lack specificity to the target RNA.

    Target Structure

    Inside cells, RNA folds into secondary structures and form complexes with ribonucleoproteins into tertiary structures. These higher order structures can prevent the ASO from hybridizing to their theoretical target sequence. So when selecting a suitable target sequence, the predicted secondary and tertiary structures should be considered, and in vivo binding studies should be performed to confirm target-binding.

    Nuclease Stability

    Both endonucleases and exonucleases can cause degradation in vivo. All ASOs are chemically modified to resist nuclease degradation. Phosphorothiothioate modifications along the backbone of the ASO are introduced to resist nuclease degradation.

    Secondary Structure and Dimers

    When designing the sequence, base self-pairing in the antisense oligonucleotide and formation of ASO dimers should be avoided. Both self-pairing and dimer formation can hinder the oligonucleotide’s ability to effectively bind to the target sequence.

    The GC content of ASOs should be around 60%-65%. High GC content can cause self-aggregation and reduce the availability of ASOs to the target sequence, while low GC content may decrease affinity to the target.

    Functional Motifs

    The motifs CCAC, TCCC, ACTC, GCCA, and CTCT were associated with enhanced efficiency, while GGGG, ACTG, AAA, TAA and these motifs reduced antisense activity.

    Off-Target

    The final unmodified antisense oligonucleotide sequence should be searched for BLAST to check for any off-target hybridization.

  • Modifications permitted for individualized ASOs

    Modifications permitted for individualized ASOs

    ASOs usually consist of 15-20 nucleotides that are complementary to the target mRNA sequence. Chemically modified ribonucleotides are used to protect against nuclease degradation, improve target affinity and delivery to the intended target/tissue/organ.

    In the FDA’s Guidance, individualized ASOs are limited to well-characterized antisense chemical classes where substantial nonclinical information and clinical experience are available. These include single-stranded phosphorothioate or mixed phosphorothioate/phosphodiester with or without 2-methoxyethyl substituted oligonucleotides (by systemic or intrathecal route), and phosphorodiamidate morpholino oligonucleotides (by systemic route).

    Oligonucleotide phosphorothioate modification is the most widely used backbone variant: one of the oxygen atoms in the phosphate backbone is replaced by sulfur, providing nuclease resistance as well as facilitating cell membrane entry.

    The oligonucleotide phosphorothioate modification.

    However, phosphorothioates show sequence-independent, but length-dependent binding to cellular proteins (heparin-binding molecules) and show up as phosphorothioate-linked thrombocytopenia (low blood platelet count).

    Increasing the number of phosphorothioate bonds in an oligo tends to lower the melting temperature (Tm) by around 0.5 °C per PS bond, thereby affecting hybridisation to the target. Introduction of too many sulfurization process during synthesis creates stereogenic α-phosphorus atoms, giving rise to diastereomers. These stereoisomers have different functional properties that can limit antisense pairing.

    For intrathecal dosing, RNA 2′-O-methylation is the only remaining RNA modification permitted by the FDA for use in individualized ASOs. RNA 2′-O-methylation is highly abundant in noncoding RNAs and occurs in the 5′ cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes.

    2′-O-Methylation.

    2′-O-methylation has the potential to affect RNAs in multiple ways as it can increase their hydrophobicity, protect them from nuclease attacks, stabilize helical structures, and affect their interactions with proteins or other RNAs.