Specificity of oligonucleotide gene therapy (OGT) agents

Abstract

Oligonucleotide gene therapy (OGT) agents (e. g. antisense, deoxyribozymes, siRNA and CRISPR/Cas) are promising therapeutic tools. Despite extensive efforts, only few OGT drugs have been approved for clinical use. Besides the problem of efficient delivery to targeted cells, hybridization specificity is a potential limitation of OGT agents. To ensure tight binding, a typical OGT agent hybridizes to the stretch of 15-25 nucleotides of a unique targeted sequence. However, hybrids of such lengths tolerate one or more mismatches under physiological conditions, the problem known as the affinity/specificity dilemma. Here, we assess the scale of this problem by analyzing OGT hybridization-dependent off-target effects (HD OTE) in vitro , in animal models and clinical studies. All OGT agents except deoxyribozymes exhibit HD OTE in vitro , with most thorough evidence of poor specificity reported for siRNA and CRISPR/Cas9. Notably, siRNA suppress non-targeted genes due to (1) the partial complementarity to mRNA 3'-untranslated regions (3'-UTR), and (2) the antisense activity of the sense strand. CRISPR/Cas9 system can cause hundreds of non-intended dsDNA breaks due to low specificity of the guide RNA, which can limit therapeutic applications of CRISPR/Cas9 by ex-vivo formats. Contribution of this effects to the observed in vivo toxicity of OGT agents is unclear and requires further investigation. Locked or peptide nucleic acids improve OGT nuclease resistance but not specificity. Approaches that use RNA marker dependent (conditional) activation of OGT agents may improve specificity but require additional validation in cell culture and in vivo .

Functional roles, regulatory mechanisms and theranostics applications of ncRNAs in alcohol use disorder

“…and RNA drugs (e.g., antisense oligonucleotide (ASO) drugs, RNA interference drugs, mRNA gene therapy, etc. ), are promising therapeutic tools [108]. After years of tortuous development, a total of about 43 gene therapy drugs have been approved for clinical application and more than 3,000 preclinical and clinical trials of gene therapy are under way or have been completed worldwide [109].…”

Thresholding DNA Nanomachine as a Highly Cooperative and Efficient Enzyme‐Like System for Controlled RNA Cleavage

“…Unfortunately, it is still not feasible for TNAs to surpass the clinical trials phase regarding cancer treatment. The main hurdles keeping TNA technology from clinical use are unsuccessful systematic intracellular delivery, low stability, and difficulty in the target‐molecule choice, which leads to low selectivity and off‐target effect [4–6] . Advances in nucleic acid nanotechnology have been made to overcome these problems by developing multivalent, multifunctional, and highly programmable nucleic acid nanostructures [7] …”

“…The main hurdles keeping TNA technology from clinical use are unsuccessful systematic intracellular delivery, low stability, and difficulty in the target-molecule choice, which leads to low selectivity and off-target effect. [4][5][6] Advances in nucleic acid nanotechnology have been made to overcome these problems by developing multivalent, multifunctional, and highly programmable nucleic acid nanostructures. [7] Nucleic acid nanotechnology has demonstrated the ability to carry therapeutic cargo (peptides, small molecules, etc.)…”

Cleaving Folded RNA with DNAzyme Agents

“…[6,7,10] Moreover, in contrast to all other OGT agents, Dz rarely exhibit nonspecific cleavage of unrelated RNA in a transcriptome. [11] This high specificity is a consequence of the separation of two RNA binding Arms (Arm 1 and 2) by the catalytic core: the overall recognition of a 15-20-nucleotide (nt) target region is achieved by binding two arms to the RNA target, each of which is short enough to be destabilized by a single-base mismatch, the effect best documented for binary (split) probes. [12] As a downside, two short RNA-binding arms bind the RNA weaker than a single uninterrupted probe.…”