Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS

Abstract

Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.

Preclinical evaluation of WVE-004, an investigational stereopure oligonucleotide for the treatment of C9orf72-associated ALS or FTD

“…Overall, these data indicate that WVE-004 does not induce cytotoxicity and that it induced relatively low cytokine production compared with the positive control in vitro at the concentrations tested in this assay. WVE-004’s impact on PBMC cytokine secretion is consistent with our prior report that PN-modified oligonucleotides are not pro-inflammatory and may be less inflammatory than a PS-modified counterpart, 45 which may be critical if the C9orf72 -repeat expansion leads to a pro-inflammatory state. 34 , 35 …”

“…It contains a combination of stereopure PS and PN linkages, which we have previously shown can improve potency, distribution, and durability compared with similar PS-modified antisense oligonucleotides in the CNS. 45 Herein, we reinforce these findings by showing that WVE-004 can lead to sustained reductions in V3 transcripts and poly-GP in the spinal cord and cortex for at least 6 months. To our knowledge, this long-term effect with a relatively low dose is unprecedented in the CNS of mouse models.…”

“… 7 Subsequent data have shown that the potency, CNS delivery, and duration of activity of stereopure antisense oligonucleotides can be improved by introducing phosphoryl guanidine-containing backbone linkages (phosphoryl guanidine [PN] chemistry) into their backbone. 45 This modification, like the more-familiar phosphorothioate (PS) modification, replaces the phosphodiester (PO) bond with a nitrogen-containing moiety in the oligonucleotide backbone, creating a chiral configuration. With PS modification, the oxygen is replaced by sulfur; with PN modification, the oxygen is replaced by a phosphoryl-guanidine moiety.…”

Synthesis of Oligonucleotides Carrying Inter-nucleotide N-(Benzoazole)-phosphoramide Moieties

“…In addition, it has been shown that the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics. Incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase the potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS)- and phosphodiester (PO)-based molecules …”

“…Incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase the potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereo-pure phosphorothioate (PS)-and phosphodiester (PO)-based molecules. 16 Recently, the advantages of PN applications in an allelespecific real-time PCR analysis have been shown. The introduction of phosphorylguanidine modification(s) leads to a significant increase in the selectivity and a decrease in the concentration detection limit of the mutant DNA.…”