ASO targeting RBM3 temperature‐controlled poison exon splicing prevents neurodegeneration in vivo

Abstract

Neurodegenerative diseases are increasingly prevalent in the aging population, yet no disease-modifying treatments are currently available. Increasing the expression of the cold-shock protein RBM3 through therapeutic hypothermia is remarkably neuroprotective. However, systemic cooling poses a health risk, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon within the RBM3 gene that is solely responsible for its cold-induced expression. Genetic removal or antisense oligonucleotide (ASO)-mediated manipulation of this exon yields high RBM3 levels independent of cooling. Notably, a single administration of ASO to exclude the poison exon, using FDA-approved chemistry, results in long-lasting increased RBM3 expression in mouse brains. In prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss and spongiosis despite high levels of disease-associated prion protein.Our promising results in mice support the possibility that RBM3inducing ASOs might also deliver neuroprotection in humans in conditions ranging from acute brain injury to Alzheimer's disease.

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

“…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).…”

“…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.…”

Alternative 3′UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner

“…Interestingly, our data do suggest a previously unknown mechanism for APA regulation in T cell via induced expression of RBM3 early upon T cell activation. RBM3 is a stress-induced protein that has been implicated in cytokine expression in innate lymphoid cells 39 , and has neural protective activity 40 . We show here that expression of RBM3 mRNA and protein are markedly enhanced early after T cell activation and this correlates with 3′UTR shortening of genes that have an RBM3 binding motif immediately overlapping the distal PAS site.…”

Stabilizing mammalian RNA thermometer confers neuroprotection in subarachnoid hemorrhage

“…Using the neuroprotective and cold-induced RBM3 as an example, we characterize a specific rG4 in detail. We find that stabilizing rG4s located proximal to the splice sites of RBM3’s cold-repressed poison exon 46,47 , either through low temperature or high potassium concentration, promotes exon skipping, thereby increasing RBM3 expression by evading nonsense-mediated decay (NMD) 46,47 . Importantly, increasing intracellular potassium level via a potassium channel blocker (4-aminopyridine, 4-AP) stabilizes rG4s and confers RBM3-dependent neuroprotection in a subarachnoid hemorrhage mouse model.…”