Dual-isoform hUBE3A gene transfer improves behavioral and seizure outcomes in Angelman syndrome model mice
Matthew C. Judson, Charles Shyng, Jeremy M. Simon, Courtney R. Davis, A. Mattijs Punt, Mirabel T. Salmon, Noah W. Miller, Kimberly D. Ritola, Ype Elgersma, David G. Amaral, Steven J. Gray, Benjamin D. Philpot
Matthew C. Judson, Charles Shyng, Jeremy M. Simon, Courtney R. Davis, A. Mattijs Punt, Mirabel T. Salmon, Noah W. Miller, Kimberly D. Ritola, Ype Elgersma, David G. Amaral, Steven J. Gray, Benjamin D. Philpot
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Research Article Neuroscience Therapeutics

Dual-isoform hUBE3A gene transfer improves behavioral and seizure outcomes in Angelman syndrome model mice

Abstract

Loss of the maternal UBE3A allele causes Angelman syndrome (AS), a debilitating neurodevelopmental disorder. Here, we devised an AS treatment strategy based on reinstating dual-isoform expression of human UBE3A (hUBE3A) in the developing brain. Kozak sequence engineering of our codon-optimized vector (hUBE3Aopt) enabled translation of both short and long hUBE3A protein isoforms at a near-endogenous 3:1 (short/long) ratio, a feature that could help to support optimal therapeutic outcomes. To model widespread brain delivery and early postnatal onset of hUBE3A expression, we packaged the hUBE3Aopt vector into PHP.B capsids and performed intracerebroventricular injections in neonates. This treatment significantly improved motor learning and innate behaviors in AS mice, and it rendered them resilient to epileptogenesis and associated hippocampal neuropathologies induced by seizure kindling. hUBE3A overexpression occurred frequently in the hippocampus but was uncommon in the neocortex and other major brain structures; furthermore, it did not correlate with behavioral performance. Our results demonstrate the feasibility, tolerability, and therapeutic potential for dual-isoform hUBE3A gene transfer in the treatment of AS.

Authors

Matthew C. Judson, Charles Shyng, Jeremy M. Simon, Courtney R. Davis, A. Mattijs Punt, Mirabel T. Salmon, Noah W. Miller, Kimberly D. Ritola, Ype Elgersma, David G. Amaral, Steven J. Gray, Benjamin D. Philpot

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Figure 2

PHP. B/hUBE3Aopt yields traceable, dual-isoform hUBE3A expression.

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PHP. B/hUBE3Aopt yields traceable, dual-isoform hUBE3A expression. (A) P...
(A) PHP.B/hUBE3Aopt construct for expressing long (isoform 3) and short (isoform 1) isoforms of hUBE3A under control of a human synapsin promoter (hSYN). (B) Top: multiple sequence alignment of hUBE3Aopt with human (hUBE3A), NHP (moUBE3A), and mouse (mUbe3a) transcripts. Darker shades of red indicate greater sequence divergence. Green lines overlay hUBE3Aopt and mUbe3a amplicons in ddPCR assays. Bottom left: mean ± SEM transcript values (normalized to Gapdh) for hUBE3Aopt (top) and mUbe3a (bottom) in the brains of adult WT and AS mice following neonatal ICV administration of 1 μL of either vehicle or 1.6 × 1014 vg/mL PHP.B/hUBE3Aopt. Bottom right: representative HCR in situ hybridization labeling for hUBE3Aopt transcripts (green) in the neocortex and hippocampus of 1-month-old WT and AS + AAV mice (n = 3). Higher magnification depicts labeling in Rbfox3+ (magenta) neocortical neurons; arrowheads indicate nuclei of putative glia. Scale bars by level of magnification: 350 μm (low) and 5 μm (high). (C) Top: Western blotting of whole-brain lysates using a UBE3A antibody that binds mouse and human UBE3A equally, irrespective of isoform. Asterisks indicate the position of long (red) and short (black) UBE3A isoforms. Coomassie staining shows total protein loading. AS + AAV mice were administered 1 μL of 1.6 × 1014 vg/mL PHP.B/hUBE3Aopt as neonates. Homozygous mISO3-KO mice completely lack expression of short UBE3A, while mice with a maternally inherited deletion of isoform 3 (mat. ISO3-KO) lack neuronal expression of short UBE3A. Bottom: mean ± SEM fractional values of long and short UBE3A expression (left) and short/long isoform ratios (right). (D) Immunoblot demonstrating catalytic activity toward self (autoubiquitination) and RING1B (target ubiquitination) of different UBE3A constructs in an E. coli–based ubiquitination assay. Higher molecular weight ubiquitin-specific modifications were indicative of catalytic activity for WT UBE3A and hUBE3Aopt samples, but not samples expressing catalytically impaired UBE3AC840S. See supplemental material for unedited blots.