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

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.


In situ hybridization
Hybridization Chain Reaction (HCR, Molecular Instruments, Los Angeles, CA) was used to visualize multiple cellular RNA targets in fresh frozen tissues. The Rbfox3 probe set was designed against transcript variant 1 (NM_001039167.1). A custom hUBE3Aopt probe set was designed against a target region spanning 223-2434 bp of the hUBE3Aopt vector coding sequence (Table S1). Briefly, brains were rapidly harvested via dissection and snap-frozen in dry ice-cooled isopentane prior to sectioning on a cryostat to a thickness of 20 μm. HCR was performed according to manufacturer specifications.

Western blotting analysis
Approximately 15 μg of total protein lysate, extracted from one whole cerebral hemisphere per sample, was loaded onto a 4-15% polyacrylamide gel and separated by SDS-PAGE.
Electrophoresed proteins were transferred onto nitrocellulose membranes, which were subsequently blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE, 927-50000) for 1 h at room temperature. Blocked membranes were incubated at 4 °C overnight with primary antibodies diluted in a 1:1 solution of blocking buffer and Tris-buffered saline containing 0.1% Tween-20 (TBST). Primary antibodies included mouse anti-UBE3A (Sigma, 1404508, RRID:AB_10740376), diluted 1:1,000. Membranes were then washed repeatedly with TBST prior to incubation for 1 h at room temperature with secondary antibodies prepared in the same diluent as the primary antibodies. Secondary antibodies included 800cw donkey anti-mouse IgG (LI-COR, 926-32212), diluted 1:7,500. At the end of the incubation, membranes were washed repeatedly with TBST, followed by 3 final washes with TBS, immediately preceding analysis of infrared fluorescence using an Odyssey CLx imager (LI-COR).

E. coli in vivo ubiquitination assay
The intrinsic catalytic activity of hUBE3Aopt was assessed with a previously established E. coli-

Behavioral testing and analysis
Open field: Locomotor activity in a novel environment was assessed in a photocell-equipped automated chamber (41 cm × 41 cm × 30 cm; Versamax system, Accuscan Instruments).
Activity chambers were contained inside sound-attenuating boxes equipped with ceilingmounted lights and fans. Total distance traveled and rearing movements were recorded over the course of a 1 h trial.
Marble burying: Mice were tested in a clean cage located in a sound-attenuating chamber with ceiling light and fan. The cage contained 3 L of corncob bedding (Andersons Lab, 1/8 in diameter, irradiated), approximately 5 cm deep. 20 black glass marbles (14 mm diameter) were arranged on top of the bedding in an equidistant 5 × 4 grid. Animals were given access to the marbles for 30 min. At the end of the trial, mice were carefully removed from the cage, and the number of buried marbles (i.e., at least 50% covered by bedding) was recorded. In addition, overhead photographs of the testing arena were taken at the start and end of each trial, from which the percentage of marble area obscured by bedding was digitally computed using ImageJ software (RRID:SCR_003070) (4).
Rotarod: Mice were tested for balance and motor coordination on an accelerating rotarod (UgoBasile, Stoelting Co., Wood Dale, Il). Revolutions per minute (rpm) were set at an initial value of 3, with a progressive increase to a maximum of 30 rpm across 5 min, the maximum trial length. 3 consecutive trials were administered to each mouse during the acquisition session. 48 hr later, two additional trials were conducted during the retest session. Intertrial intervals were 3-5 min. Latency to fall off the rotarod, or until the occurrence three consecutive wrapping/passive rotations, was recorded.
Fear conditioning: The Near-Infrared image tracking system (MED Associates, Burlington, VT) was used to conduct the fear-conditioning procedure across 3 days. On the first day, the training session, mice were placed in the test chamber, contained in a sound-attenuating box, and allowed to explore for 2 min to establish baseline levels of mobility and immobility. Mice were then exposed to a 30-s tone (80 dB), followed by a 2-s foot shock (0.4 mA). Mice received 2 additional shock-tone pairings, with 80 s intertrial intervals. Increased freezing during successive 30-s tone presentations was interpreted as learning of the shock-tone association. Contextdependent learning was evaluated on the second day of testing. Mice were placed back into the original test chamber, and levels of freezing (immobility) were determined across a 5-min session. On the third day of testing, mice were evaluated for associative learning of the auditory cue in a final 5-min session, which took place in chambers modified to obscure the original testing context: plexiglass inserts were added to change the wall and floor surface, and a novel odor (vanilla flavoring) was added to the sound-attenuating box. After 2 min of habituation to the novel context, the acoustic stimulus was presented for a 3-min period, wherein levels of freezing were analyzed.
Nest building: Mice were single-housed for a period of 3 to 5 days before the start of the experiment. On day 1, used nesting material provided by the animal care facility was replaced with 11 ± 1 g of compressed extra-thick blot filter paper (Bio-Rad, 1703966), cut into 8 evenly sized rectangles. The amount of paper not incorporated into a nest was weighed and noted each day, for 5 consecutive days. On the final day of the assay, mice were carefully removed from their cages and overhead photographs of their nests were taken for post hoc analysis of nest quality, assessed according to a 5-point ordinal scale adapted from a previous study (5)  Mice: Mice were anesthetized with sodium pentobarbital (60 mg/kg) prior to transcardial perfusion with PBS, immediately followed by phosphate-buffered 4% paraformaldehyde, pH 7.3.
Immunofluorescent staining: Brain sections were rinsed several times in PBS before blocking in PBS plus 5% normal goat serum and 0.2% Triton X-100 (NGST) for 1 h at room temperature. Imaging: Images of immunofluorescently labeled brain sections were acquired with a Zeiss LSM 710 confocal microscope equipped with ZEN imaging Software (Zeiss, RRID:SCR_013672).