Control of backbone chemistry and chirality boost oligonucleotide splice switching activity

Abstract

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.

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

“…We have previously reported a method to generate oligonucleotides containing stereopure PN linkages based on (1,3-dimethylimidazolidin-2-ylidene) phosphoramidates (Figure 1A Supplementary Figure S1 ) with 2′- O -methyl (2′-OMe) and 2′-deoxyfluoro (2′-F) ribose modifications, and we have applied these methods to the synthesis and evaluation of splice switching oligonucleotides ( 9 ). To apply this chemistry to antisense oligonucleotides that promote RNase H-mediated RNA degradation, we expanded on this capability to incorporate additional 2′-ribose modifications, such as 2′- O -methoxyethyl (2′-MOE).…”

“…To investigate whether the presence of PN-1 linkages in antisense oligonucleotides impacts these parameters, we performed additional in vivo assessments in wild-type mice. Our previous work on splice-switching oligonucleotides indicated that PN chemistry can improve oligonucleotide distribution to muscle in vivo upon systemic administration ( 9 ). To assess distribution in CNS, we measured oligonucleotide concentrations and Malat1 expression levels in the spinal cord and cortex 4-weeks after a single 100 μg ICV injection.…”

“…Towards this end, we synthesized stereopure PN linkages and their derivatives ( Supplementary Figure S1 ) and successfully synthesized chimeric PS/PN or PS/PN/PO stereopure backbone-containing oligonucleotides (Figure 1 ). We recently applied this backbone chemistry to splice switching oligonucleotides in muscle ( 9 ) and found that chimeric oligonucleotides, with a stereopure PN/PS/PO backbone, yielded profound phenotypic benefits over comparable stereopure PS/PO molecules, with activity gains deriving at least in part from improved pharmacology. We have also applied this backbone chemistry to our RNA editing oligonucleotides (Monian et al.…”

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

“…These oligonucleotides have been shown to have markedly improved pharmacology and efficacy compared to PS-derived oligonucleotides. 15 In T h i s c o n t e n t i s 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 stereo-pure phosphorothioate (PS)-and phosphodiester (PO)-based molecules.…”

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

“…It was synthesized as described previously. 56 The NTC used in this work is a stereorandom PS-modified oligonucleotide with the same 2′-ribose modification pattern as WVE-004 (5′-CCTTCCCTGAAGGTTCCUCC-3′).…”