An orthogonal dual-CRISPR epigenetic platform for paternal UBE3A reactivation
GeCure Solutions is developing and functionally validating a platform designed to restore expression of the silenced paternal UBE3A allele in Angelman syndrome through two complementary mechanisms operating simultaneously — targeted epigenetic reactivation of the paternal UBE3A promoter and independent attenuation of antisense transcription within the UBE3A-ATS domain.
Scientific aim
Two parallel, mechanistically distinct modules — acting together, independently
Unlike concepts based on internally regulated feedback circuits or complex autonomous epigenetic timers, this approach employs two parallel modules that act concurrently but independently. The design avoids reliance on dosage-control switches and does not require an internally encoded molecular trigger to transition between activation and maintenance phases.
Both modules target complementary determinants of paternal UBE3A silencing. The platform minimizes the probability of direct molecular competition or cross-talk by employing orthogonal CRISPR systems with distinct protospacer adjacent motif (PAM) recognition mechanisms and independent guide RNA scaffolds.
Background: why paternal UBE3A?
Angelman syndrome (AS) is a neurodevelopmental disorder characterized by developmental delay, absent or severely impaired speech, ataxia, epilepsy, and a distinct behavioral phenotype. It results from loss of functional maternal expression of UBE3A in neurons. In the CNS, the paternal UBE3A allele remains epigenetically silenced through genomic imprinting mediated by continuous transcription of the long non-coding antisense transcript UBE3A-ATS (SNHG14), which drives downstream transcriptional collision.
Approximately 70% of patients carry a large maternal deletion of the 15q11.2-q13.1 locus, making reactivation of the single remaining paternal UBE3A allele a rational therapeutic strategy for the majority of affected individuals. Current approaches — antisense oligonucleotides (ASOs), RNA editing, and AAV-mediated gene replacement — remain limited by vector delivery, host immunogenicity, precise dosage control, and the need for repeated intrathecal administrations. Because both insufficient and excessive UBE3A expression are linked to neurological dysfunction, strategies that restore physiological levels of endogenous UBE3A remain an unmet clinical need.
The dual-vector co-transduction framework
Two autonomous modules function simultaneously but independently within a dual-vector co-transduction framework.
Epigenetic reactivation axis
An ultra-compact dCasMINI (engineered Cas12f) platform fused to a minimal demethylation effector and driven by the neuronal hSyn promoter. Its ~1.5 kb coding sequence eases packaging constraints and enables compact CNS-suitable constructs. The module induces localized chromatin remodeling and epigenetic unlocking of the paternal UBE3A core promoter, lowering the transcriptional barrier that maintains paternal silencing.
Antisense attenuation axis
A constitutively active module that attenuates transcriptional interference from UBE3A-ATS in parallel with Module 1 — no feedback trigger required. It targets transcriptionally permissive intronic regions within the SNORD115 cluster, avoiding disruption of upstream imprinting architecture and minimizing risk to the SNORD116 locus linked to Prader–Willi syndrome. The rationale is consistent with antisense approaches such as the intrathecal candidate GTX-102.
Central hypothesis
The simultaneous deployment of two orthogonal CRISPR-based modules — one promoting targeted epigenetic reactivation of the paternal UBE3A promoter and another inducing localized antisense transcriptional attenuation — will achieve a more robust and durable restoration of endogenous UBE3A expression within a physiological range than either intervention alone, while preserving the structural integrity of the 15q11.2-q13.1 imprinting domain and minimizing disruption of essential upstream regulatory elements.
Six specific aims
Each aim delivers a defined experimental output — from iPSC neuronal model validation through in vivo stability and functional network rescue.
Directed neurogenesis of an AS iPSC deletion model
Differentiate human iPSCs (line 409B2 carrying a 2 Mb maternal deletion) into mature neurons and validate their phenotype — a controlled in vitro model reflecting imprinting-dependent regulation of paternal UBE3A vs. wildtype.
Design and assembly of the orthogonal dual-CRISPR platform
Clone Module 1 (pAAV-hSyn-dCasMINI-XTEN-TET1-CD with a U6-gRNA1 cassette targeting the UBE3A promoter) and construct the orthogonal Module 2 (pAAV-hSyn-dSaCas9 with a U6-gRNA2 cassette targeting SNORD115 introns).
Quantitative profiling of UBE3A restoration dynamics
Evaluate the temporal kinetics of UBE3A-ATS attenuation and paternal UBE3A mRNA and protein expression over a 30-day period post-delivery via RT-qPCR and Western blot, for individual and combined module deployment.
Epigenetic mechanism and locus safety
Map promoter demethylation with bisulfite sequencing (Methyl-Seq) and confirm preservation of SNORD116 cluster transcript levels — ensuring structural safety of the imprinting domain and excluding Prader–Willi-associated off-target defects.
In vivo stability and integration
Assess long-term persistence, biocompatibility, and transcriptional stability of the human dual-CRISPR system following stereotaxic transplantation of treated neurons into the somatosensory cortex of NOD scid gamma (NSG) mice over 3 months.
Functional neuronal network rescue via MEA
Evaluate recovery of extracellular network activity by multi-electrode array (MEA) recordings — firing rates, burst dynamics, and network synchrony indices — in treated vs. untreated neuronal cultures.
Expected outcomes and impact
Completion of this project will characterize a new class of parallel, self-sustaining epigenetic therapies for imprinting disorders. It will evaluate whether the simultaneous combination of targeted epigenetic editing and transcriptional attenuation can maximize endogenous gene reactivation in post-mitotic human neurons while maintaining safety within a complex genomic domain.
This dual-vector orthogonal approach may represent a generalizable framework for treating pathologies driven by genomic imprinting asymmetries, extending beyond Angelman syndrome.
Scientific collaborations
An expanding network of international partners provides expertise, specialized technologies, and access to disease-specific research models.
ClearCut Biolabs (Max Planck Institute, Germany)
Access to the Super UniGuide platform for CRISPR guide optimization at challenging genomic targets, and to engineered 409B2 iPSC lines carrying either a 2 Mb maternal deletion of 15q11.2–q13.1 or paternal uniparental disomy (UPD15). Cell line access is coordinated via the RIKEN Cell Bank under standard material transfer procedures.
Department of Neurology, Aarhus University Hospital (Denmark)
Development and optimization of focused ultrasound (FUS)-mediated blood–brain barrier opening for targeted CNS delivery of gene therapies — supporting future translational development of brain-targeted therapies.
Danylo Halytsky Lviv National Medical University (Ukraine)
Memorandum of Understanding providing access to the university's accredited animal research facility (vivarium) and the infrastructure required for future preclinical in vivo studies in accordance with institutional and ethical regulations.
Project timeline and FAST funding scope
A 36-month translational development program aimed at advancing the platform toward the preclinical testing stage. FAST grant funding supports the first 12 months — generating the proof-of-concept and feasibility data needed to move the platform into subsequent in vivo and preclinical development.
Year 1 · FAST-funded
Proof of concept
iPSC neuronal models, module construction, in vitro efficacy, mechanism and locus-safety data, MEA-based functional rescue.
Years 2–3
In vivo and delivery
CNS delivery optimization, in vivo validation, biodistribution, durability, and safety studies toward preclinical testing readiness.
Long-term goal
Disease-modifying therapy
A strategy intended for future application in patients with Angelman syndrome by restoring endogenous paternal UBE3A expression in neurons.
Year 1 activities supported by FAST funding
- 1Establishment and validation of human AS iPSC-derived neuronal models, including differentiation of 409B2 iPSCs and confirmation of paternal UBE3A silencing.
- 2Design, construction, and optimization of the two orthogonal CRISPR modules — paternal UBE3A promoter reactivation and UBE3A-ATS attenuation.
- 3Experimental evaluation of individual and combined module activity in human neuronal cultures: UBE3A mRNA restoration, UBE3A protein expression, and UBE3A-ATS attenuation.
- 4Initial assessment of epigenetic mechanism and locus safety — promoter methylation analysis and SNORD116 transcript preservation.
- 5Functional in vitro assessment of neuronal rescue with MEA recordings and complementary molecular assays.
- 6Generation of a comprehensive data package to support Years 2 and 3: CNS delivery optimization, in vivo validation, safety evaluation, and preclinical testing readiness.
Begin with feasibility, not a full development commitment
A feasibility assessment can help you decide whether the earliest assumptions justify laboratory work.
You can begin with a document review. You do not need to commit to a research project.