ASO targeting temperature-controlledRBM3poison exon splicing prevents neurodegeneration in vivo

Abstract

Neurodegenerative diseases become increasingly prevalent in the aging population and currently no cure is available. Increasing expression of the cold-shock protein RBM3 through therapeutic hypothermia is remarkably neuroprotective, but hypothermia poses a health risk itself, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon that is solely responsible for temperature-controlled RBM3 expression. Genetic removal or ASO-mediated manipulation of this exon yields high RBM3 levels independent of cooling. Remarkably, a single administration of ASO, using FDA-approved chemistry, results in long-lasting increase of RBM3 expression and remarkable neuroprotection, with prevention of neuronal loss and spongiosis in prion-diseased mice. RBM3-inducing ASOs could thus broadly deliver protection in humans in conditions from acute brain injury to Alzheimers disease.

HNRNPH1 regulates the neuroprotective cold‐shock protein RBM3 expression through poison exon exclusion

“…In line with this, we report cooling‐dependent PE exclusion as a level of regulation governing RBM3 induction. Interestingly, this alternative splicing regulation is conserved in different cell types and between human and mouse (Preußner et al , 2023). Depletion of HNRNPH1 using different methods disrupted the alternative splicing control around RBM3 PE in endogenous RBM3 mRNA and in an externally introduced minigene.…”

“…While small molecules affecting global splicing activity have been applied to cancer treatments (Agrawal et al, 2018), modulating specific splicing events by targeting cis-acting elements using splice-switching antisense oligonucleotides (ASOs) show therapeutic benefit in several genetic diseases, including spinal muscular atrophy and Duchenne muscular dystrophy (Havens & Hastings, 2016). Most recently, ASOs designed to repress RBM3 PE inclusion successfully upregulated RBM3 expression, without cooling, leading to marked neuroprotection in mice with prion neurodegeneration (Preußner et al, 2023), consistent with the protective effects of inducing RBM3 by hypothermia (Peretti et al, 2015) or other means (Peretti et al, 2021). Beyond ASO technology, the past two decades have witnessed the development of splicing control using bifunctional oligonucleotides, consisting of an antisense domain complementary to the mRNA region close to the splice site, and a tail domain recruiting RBPs to promote exon inclusion or exclusion as an alternative approach (Skordis et al, 2003;Zhou, 2022).…”

HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion

“…In line with this, we report cooling-dependent PE exclusion as a level of regulation governing RBM3 induction. Interestingly, this alternative splicing regulation is conserved in different cell types and between human and mouse (Preussner et al, 2022). Depletion of HNRNPH1 using different methods disrupted the alternative splicing control around RBM3 PE in endogenous RBM3 mRNA and in an externally introduced minigene.…”

“…Although small molecules affecting global splicing activity has been applied to cancer treatments (Agrawal et al, 2018), modulating specific splicing events by targeting cis-acting elements using splice-switching antisense oligonucleotides (ASOs) has confirmed its therapeutic benefits in several genetic diseases, including spinal muscular atrophy and Duchenne muscular dystrophy (Havens and Hastings, 2016). Repressing PE inclusion in RBM3 using ASOs successfully upregulates RBM3 expression for neuroprotection in mice with prion neurodegeneration (Preussner et al, 2022). The past two decades have witnessed the development of splicing control using bifunctional oligonucleotides, consisting of an antisense domain complementary to the mRNA region close to the splice site, and a tail domain recruiting RBPs to promote exon inclusion or exclusion (Skordis et al, 2003; Zhou, 2022).…”

RAN translation of C9orf72-related dipeptide repeat proteins in zebrafish recapitulates hallmarks of amyotrophic lateral sclerosis and identifies hypothermia as a therapeutic strategy

“…Lastly, a recent paper identified that RBM3 pre-mRNA undergoes alternative splicing that is sensitive to changes in temperature. A previously uncharacterized exon (exon3a) is not included in the mRNA at 34 o C, however exon3a-containing mRNA isoforms are present at 38 o C. Furthermore, antisense oligonucleotide administration targeting exon3a results in a sustained increase in RBM3 expression as well as neuroprotection in a mouse model of prion disease 35 . These mechanisms could be further explored in the context of C9orf72-ALS/FTD.…”