Authors :
Presenting Author: Perry Spratt, PhD – Regel Therapeutics
Olivia Stevenson, BS – Regel Therapeutics
Jaclyn Essig, PhD – Regel Therapeutics
Andrew Field, PhD – Regel Therapeutics
Nicholas Trojanowski, PhD – Regel Therapeutics
Joshua Reiser, MS – Regel Therapeutics
Tasneem Rinvee, BS – Regel Therapeutics
Michael Faundo, MSc – Regel Therapeutics
Yan Hung, BS – Regel Therapeutics
Ashwini Shinde, BS – Regel Therapeutics
Navneet Matharu, PhD – Regel Therapeutics
Jordane Dimidschstein, PhD – Regel Therapeutics
Kathryn Allaway, PhD – Regel Therapeutics
Rationale:
Many genes implicated in monogenic neurodevelopmental disorders are selectively expressed in neurons of the central nervous system (CNS), with minimal transcription in peripheral tissues. Developing strategies that achieve neuron-restricted rescue of gene expression has the potential to significantly improve the efficacy and safety of gene modulation therapies for CNS disorders. To explore this, we focused on SCN2A haploinsufficiency, a genetic cause of autism spectrum disorder, developmental delay, and epilepsy. SCN2A, which encodes the voltage‑gated sodium channel Nav1.2, is highly enriched in CNS neurons, making it a target well suited to validate this approach.
Methods:
A rational approach was used to engineer a suite of regulatory elements (REs) to drive neuron-specific expression within the CNS while minimizing expression in peripheral tissues. The expression profiles of these REs were characterized in mice following intravenous injection of AAV.PHPeB vectors carrying fluorescent reporters under the control of each RE. These efforts resulted in the CNS2-V4.1 RE, which features a suitable profile for SCN2A gene modulation therapy. In parallel, to advance a therapeutic strategy for SCN2A haploinsufficiency, a dCas9-VP64 module was developed and validated in cell lines for its ability to upregulate SCN2A and its protein product, Nav1.2. This payload, driven by CNS2-V4.1, was then evaluated in vivo in mice.
Results:
In vivo screening using a reporter construct identified a set of REs with strong, neuron-specific activity and minimal to no detectable expression in non-neuronal CNS cell types or peripheral tissues following systemic (IV) AAV administration in mice. These REs exhibited a range of expression profiles, from broad pan-neuronal expression to regionally restricted activity in structures such as the cortex and cerebellum. Among these, CNS2-V4.1 was selected as the optimal candidate to restore functional gene expression in SCN2A-haploinsufficiency disorders based on its strong and broad neuronal expression across the brain and low off-target expression in peripheral organs, including the liver. In vitro studies led to the identification of a dCas9-VP64 module capable of significantly upregulating SCN2A in neuroblastoma cells. When combined with the CNS2-V4.1 promoter and packaged in an AAV9 vector, this therapeutic module achieved robust and widespread expression in vivo in the mouse brain.
Conclusions:
Together, these studies led to the development of a suite of REs suitable for therapeutic applications across a range of rare neurodevelopmental disorders. CNS2-V4.1 emerged as the lead candidate for driving expression of a dCas9-VP64 payload to address SCN2A haploinsufficiency, with both in vitro and in vivo data supporting its further development as a therapeutic vector.
Funding:
Regel Therapeutics, Inc.