Therapeutic Oligonucleotides: State of the Art

Abstract

Oligonucleotides (ONs) can interfere with biomolecules representing the entire extended central dogma. Antisense gapmer, steric block, splice-switching ONs, and short interfering RNA drugs have been successfully developed. Moreover, antagomirs (antimicroRNAs), microRNA mimics, aptamers, DNA decoys, DNAzymes, synthetic guide strands for CRISPR/Cas, and innate immunity-stimulating ONs are all in clinical trials. DNA-targeting, triplex-forming ONs and strand-invading ONs have made their mark on drug development research, but not yet as medicines. Both design and synthetic nucleic acid chemistry are crucial for achieving biologically active ONs. The dominating modifications are phosphorothioate linkages, base methylation, and numerous 2′-substitutions in the furanose ring, such as 2′-fluoro, O-methyl, or methoxyethyl. Locked nucleic acid and constrained ethyl, a related variant, are bridged forms where the 2′-oxygen connects to the 4′-carbon in the sugar. Phosphorodiamidate morpholino oligomers, carrying a modified heterocyclic backbone ring, have also been commercialized. Delivery remains a major obstacle, but systemic administration and intrathecal infusion are used for treatment of the liver and brain, respectively.

Antisense Oligonucleotide Reverses Leukodystrophy in Canavan Disease Mice

“…Mice heterozygous for the Aspa nonsense mutation Aspa nur7 were obtained from the Jackson Laboratory (JAX:008607; Bar Harbor, ME), maintained on a C57BL/6J background, and crossed to generate Aspa nur7/nur7 CD mice and age‐matched Aspa WT/WT control mice (WT). An LNA‐ASO designed to knockdown Nat8l expression (Nat8l gapmer, nucleotide sequence GGCGTAGAGCAGTTGG), and a negative control LNA‐ASO without known eukaryote targets (control gapmer, nucleotide sequence AACACGTCTATACGC), were from Qiagen (Germantown, MD). Both gapmers were constructed with a phosphorothioate backbone and 2′‐O‐methoxyethyl‐derivatized sugar modifications .…”

“…An LNA‐ASO designed to knockdown Nat8l expression (Nat8l gapmer, nucleotide sequence GGCGTAGAGCAGTTGG), and a negative control LNA‐ASO without known eukaryote targets (control gapmer, nucleotide sequence AACACGTCTATACGC), were from Qiagen (Germantown, MD). Both gapmers were constructed with a phosphorothioate backbone and 2′‐O‐methoxyethyl‐derivatized sugar modifications . Nat8l or control gapmer (0.5nmol in 5μl of artificial cerebrospinal fluid) was infused into the cisterna magna of 2‐month‐old CD or WT mice at 1μl/min under isoflurane anesthesia.…”

“…4,5 The brain concentration of NAA ([NAA B ]) is markedly elevated in CD, 2,4 and also in aspartoacylasedeficient CD mice. [6][7][8][9] We now report that lowering [NAA B ] in young-adult CD mice by administration into cisterna magna of a locked nucleic acid antisense oligonucleotide (LNA-ASO, or gapmer) 10 to knockdown expression of the neuronal NAA-synthesizing enzyme N-acetyltransferase 8-like (Nat8l) 11 reverses ataxia, Purkinje cell dendritic atrophy, and cerebellar/thalamic vacuolation.…”

Copper‐Catalyzed Huisgen 1,3‐Dipolar Cycloaddition Tailored for Phosphorothioate Oligonucleotides

“…Nucleic-acid therapeutics are currently experiencing a tremendous expansion, including several recent FDA approvals of ON drugs (Smith & Zain, 2019;Stein & Castanotto, 2017). In addition, there are numerous ongoing clinical trials (Shen & Corey, 2018;Sridharan & Gogtay, 2016).…”

CRISPR-Cas-amplified urine biomarkers for multiplexed and portable cancer diagnostics

“…Even measurements of endogenous, cell-free nucleic acids are hampered by the degradation of sequences that are not protected by the nucleosome in circulation [10][11][12][13] . Chemically-modified nucleic acids which have been primarily developed as therapeutics are less resistant to systemic degradation; however, they have not been incorporated into diagnostic platforms because they cannot be amplified with conventional polymerases, nor inexpensively sequenced [14][15][16] .…”