Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats

Abstract

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an incurable neurodegenerative disorder caused by expansion of CGG repeats in the FMR1 5’UTR. The RNA containing expanded CGG repeats (rCGGexp) causes cell damage by interaction with complementary DNA, forming R-loop structures, sequestration of nuclear proteins involved in RNA metabolism and initiation of translation of polyglycine-containing protein (FMRpolyG), which forms nuclear insoluble inclusions. Here we show the therapeutic potential of short antisense oligonucleotide steric blockers (ASOs) targeting directly the rCGGexp. In nuclei of FXTAS cells ASOs affect R-loop formation and correct miRNA biogenesis and alternative splicing, indicating that nuclear proteins are released from toxic sequestration. In cytoplasm, ASOs significantly decrease the biosynthesis and accumulation of FMRpolyG. Delivery of ASO into a brain of FXTAS mouse model reduces formation of inclusions, improves motor behavior and corrects gene expression profile with marginal signs of toxicity after a few weeks from a treatment.

Trinucleotide CGG Repeat Diseases: An Expanding Field of Polyglycine Proteins?

“…In that aspect, constant efforts during the last decade to develop small pharmacological molecules specifically binding CGG RNA repeats in vitro , and that now efficiently decrease expression of the toxic FMRpolyG in cell and animal models provide exciting therapeutic hope for these disorders ( Disney et al, 2012 ; Qurashi et al, 2012 ; Tran et al, 2014 ; Yang et al, 2016 ; Green et al, 2019 ; Verma et al, 2020 ; Asamitsu et al, 2021 ; Haify et al, 2021 ; Malik et al, 2021b ; Konieczny et al, 2021 ). In parallel, major advances in antisense oligonucleotides (ASO) chemistry to reduce their toxicity and improve their biodistribution and cellular uptake open new hope to identify ASOs sequences targeting these CGG repeats to promote their RNA degradation and/or block their translation into toxic polyglycine-containing proteins, as recently reported in animal models of FXTAS ( Rodriguez et al, 2020 ; Derbis et al, 2021 ).…”

(Dys)function Follows Form: Nucleic Acid Structure, Repeat Expansion, and Disease Pathology in FMR1 Disorders

“…While the ability of the FX repeats to form secondary structures of various sorts has been known for some time, work in recent years has begun to identify ways to target these structures or the downstream consequences of these structures, so as to ameliorate their effects. For example, CCG-repeat-containing antisense oligonucleotides (ASOs) reduce R-loop formation and ameliorate some of the downstream consequences of the formation of RNA hairpins [143]. Small molecules that target CGG-RNA hairpins have also been shown to have beneficial effects in cell and mouse models of the PM [144][145][146].…”

Gene Therapies for Monogenic Autism Spectrum Disorders

“…Healthy individuals can have several CGG repeats in this region, but when this number exceeds 55, patients develop fragile X associated tremor/ataxia syndrome (FXTAS) or fragile X syndrome (FXS) when the repeat number exceeds 200. By designing an ASO that targets toxic CGG repeats in the FMR1 mRNA, the amount of disease-causing transcripts was reduced in vitro and in vivo [15] A variation of this approach is used for Angelman syndrome (AG), caused by structural variants in the 15q11-13 locus, often leading to complete loss of the UBE3A gene, other LoF mutations in UBE3A or imprinting errors leading to inactivation of the maternal copy of the gene. In healthy individuals, the paternal copy of the UBE3A gene is imprinted, leading to a maternal-specific expression, which, in the case of AS, is abolished by LoF mutations.…”