Kremer, T. | bioRxiv Science

bioRxiv2024

UBE3A reinstatement restores behavior and proteome in an Angelman Syndrome mouse model of Imprinting Defects

Angelman Syndrome (AS) is a severe neurodevelopmental disorder wionly symptomatic treatment currently available. Besides mutations within the UBE3A gene, AS is caused by deletions, imprinting center defects (mICD) or uniparental disomy of chromosome 15 (UPD). Current mouse models are Ube3a-centric and do not address expression changes of other 15q11-q13 genes on AS pathophysiology. Here, we studied a mouse line that harbors a mutation affecting the AS-PWS imprinting center, hence modeling mICD/UPD AS subtypes. mICD mice showed significant reduction in UBE3A protein, bi-allelic expression of Ube3a-ATS and Mkrn3-Snord115 gene cluster, leading to robust AS behavioral deficits and proteome alterations similar to Ube3aKO mice. Genetic UBE3A overexpression in mICD mice, mimicking therapeutic strategies that effectively activate the biallelic silenced Ube3a gene, resulted in a complete rescue of all behavioral and proteome alterations. Subsequently, treatment with an antisense oligonucleotide (ASO) to directly activate the biallelic silenced Ube3a gene in mICD mice also resulted in efficient reinstatement of UBE3A, alongside a partial rescue of behavioral phenotypes. Taken together, these findings demonstrate that UBE3A loss is the primary factor underlying AS phenotypes in the mICD/UPD mouse model, and also corroborate that UBE3A reinstatement is an attractive therapeutic strategy for mICD/UPD AS individuals.

neuroscience↗

bioRxiv2022

Angelman syndrome patient neuron screen identifies a potent and selective clinical ASO targeting UBE3A-ATS with long lasting effect in cynomolgus monkey

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A with no available treatment. Restoring UBE3A levels via downregulation of the paternally cis-acting long non-coding antisense transcript (UBE3A-ATS) is a potential disease modifying. Developing molecules targeting human UBE3A-ATS is challenging because it is expressed only in neurons and lacks animal species sequence conservation. To overcome this, we performed a library screen of locked-nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) on AS patient-derived neurons, identifying initial sequences. Further optimization led to the identification of the ASO, RO7248824, which selectively and potently reduces UBE3A-ATS, while concomitantly upregulating the UBE3A mRNA and protein. These properties held true in both human AS patient- and neurotypical-, as well as cynomolgus monkey-derived neurons. In vivo use of tool molecules in wild-type (WT) and AS Ube3am-/p+ mice, revealed a steep relationship between UBE3A-ATS knock-down and UBE3A mRNA/protein upregulation, whereby an almost 90% downregulation was needed to achieve a 50% upregulation, respectively. This relationship was confirmed in cynomolgus monkeys. Whereby, repeated lumbar intrathecal administrations of RO7248824 was well tolerated without adverse in-life effects or tissue pathology and produced a robust, long lasting (up to 3 months) paternal reactivation of UBE3A mRNA/protein across key monkey brain regions. Our results demonstrate that AS human pluripotent stem cell neurons serve as an excellent translational tool and furthermore LNA-modified ASOs exhibit excellent drug-like properties. Sustained efficacy translated to infrequent, intrathecal dosing and serves as the basis for the ongoing clinical development of RO7248824 for AS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/495066v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1c2a97eorg.highwire.dtl.DTLVardef@1059954org.highwire.dtl.DTLVardef@95835aorg.highwire.dtl.DTLVardef@1e2adea_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO From AS patient blood to a neuronal screen, identifies clinical ASO with excellent in vivo properties. (1) Patients were recruited. (2) Whereby blood was reprogrammed into hIPSC and subsequently differentiated into neurons. (3) ASOs were designed and screened on human neurons to downregulate the UBE3A-ATS likely via directed RNase H Cleavage of Nascent Transcripts. (4a) RO7248824 was identified that potently and selective reduces UBE3A-ATS, concomitantly with upregulating the UBE3A sense transcript and protein which was used for in vitro pk/pd. (4b) In parallel tool murine ASO were used demonstrate in vivo POC.(5) Pivotal nonhuman primate studies to monitor safety and predict the human dose. (6) RO7248824 is in AS clinical trial. C_FIG One Sentence SummaryFrom angelman syndrome human neuron screen to cynomolgus monkey proof of concept identifies the clinical molecule RO7248824

neuroscience↗

bioRxiv2018

Detailed structural orchestration of Lewy pathology in Parkinson’s disease as revealed by 3D multicolor STED microscopy

Post-translational modifications of alpha-synuclein (aSyn), particularly phosphorylation at Serine 129 (Ser129-p) and truncation of its C-terminus (CTT), have been implicated in Parkinsons disease (PD) pathology. To gain more insight in the relevance of Ser129-p and CTT aSyn under physiological and pathological conditions, we investigated their subcellular distribution patterns in normal aged and PD brains using highly-selective antibodies in combination with 3D multicolor STED microscopy. We show that CTT aSyn localizes in mitochondria in PD patients and controls, whereas the organization of Ser129-p in a cytoplasmic network is strongly associated with pathology. Nigral Lewy bodies show an onion skin-like architecture, with a structured framework of Ser129-p aSyn and neurofilaments encapsulating CTT aSyn in their core, which displayed high content of proteins and lipids by label-free CARS microscopy. The subcellular phenotypes of antibody-labeled pathology identified in this study provide evidence for a crucial role of Ser129-p aSyn in Lewy body formation.

neuroscience↗