Defining the Factors That Contribute to On-Target Specificity of Antisense Oligonucleotides

Abstract

To better understand the factors that influence the activity and specificity of antisense oligonucleotides (ASOs), we designed a minigene encoding superoxide dismutase 1 (SOD-1) and cloned the minigene into vectors for T7 transcription of pre-mRNA and splicing in a nuclear extract or for stable integration in cells. We designed a series of ASOs that covered the entire mRNA and determined the binding affinities and activities of the ASOs in a cell-free system and in cells. The mRNA bound known RNA-binding proteins on predicted binding sites in the mRNA. The higher order structure of the mRNA had a significantly greater effect than the RNA-binding proteins on ASO binding affinities as the ASO activities in cells and in the cell-free systems were consistent. We identified several ASOs that exhibited off-target hybridization to the SOD-1 minigene mRNA in the cell-free system. Off-target hybridization occurred only at highly accessible unstructured sites in the mRNA and these interactions were inhibited by both the higher order structure of the mRNA and by RNA-binding proteins. The same off-target hybridization interactions were identified in cells that overexpress E. coli RNase H1. No off-target activity was observed for cells expressing only endogenous human RNase H1. Neither were these off-target heteroduplexes substrates for recombinant human RNase H1 under multiple-turnover kinetics suggesting that the endogenous enzyme functions under similar kinetic parameters in cells and in the cell-free system. These results provide a blueprint for design of more potent and more specific ASOs.

Molecular Mechanisms of Antisense Oligonucleotides

“…However, we do understand factors that influence both potency and specificity of the potential hybridization events. We know that RNA structure is a major determinant while only rarely do proteins that are bound to RNA targets alter ASO activity [22]. We know that the number of copies of target RNAs is irrelevant as is the RNA half-life, except for rapidly cleared RNAs such as cMyc [14,22,23].…”

“…We know that RNA structure is a major determinant while only rarely do proteins that are bound to RNA targets alter ASO activity [22]. We know that the number of copies of target RNAs is irrelevant as is the RNA half-life, except for rapidly cleared RNAs such as cMyc [14,22,23]. We also know that nonpromiscuous repeats are present in many pre-mRNAs and these make excellent sites for ASO activity [24].…”

“…Armed with a solid understanding of the properties of mammalian RNase H1, we have characterized the key factors that contribute to the specificity of cleavage by RNase H1 of cognate sites for DNA-like ASOs [22] and shown that RNase H1 is limiting. We have defined the rates of all the major steps in the pharmacological effects of DNA-like ASOs [11] and shown that several proteins may reduce or block the cleavage of target RNAs by competing with RNase H1 for binding to the DNA-RNA-like heteroduplex formed by the ASOs and target RNAs [12,14].…”

Integrated Safety Assessment of 2′-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers

“…11 Consequently, ASOs are ideal agents to alter a single member of closely related gene families and even mutant versus normal gene products. 12 Thus, they have proven to be ideal agents for target validation and may be optimal agents in the treatment of many diseases.…”

Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for <em>Clostridium difficile</em>

“…Candidate 25-mer ASO sequences ideally targeting accessible loop structures 43 within the 5′UTR near the start codons were chosen through predictive in silico modeling (RNAstructure v5.8; Mfold v3.6) 44,45 of mRNA transcript local secondary structures ( Figure 3). Selected antisense sequences were evaluated for intraorganismal specificity using NCBI BLAST 46 with screening of potential off-target binding near other known initiation codons.…”