Authors :
Presenting Author: Azin E Amini, PhD – Stanford University
Ursula Haditsch, PhD – Transcripta Bio
Tao Yang, PhD – Stanford
Drishti Guin, PhD – Transcripta Bio
Long Wu, PhD – Transcripta Bio
Nicole Perfito, PhD – Transcripta Bio
Nour Omar, PhD Candidate – Stanford University
Leo Andrade, BS – Stanford
Christopher Lee-Messer, MD, PhD – Stanford
Rebecca J Levy, MD, PhD – Stanford
Frank Longo, MD, PhD – Stanford
Christopher Moxham, PhD – Transcripta Bio
Juliet Knowles, MD/PhD – Stanford University
Rationale:
SYNGAP1 encodes a Ras/Rap GTPase-activating protein critical for synaptic development and plasticity. Heterozygous SYNGAP1 pathogenic variants that result in haploinsufficiency are associated with neurodevelopmental disorders including epilepsy, autism and intellectual disability. Currently, there are no approved treatments that directly target SYNGAP1 haploinsufficiency or the underlying synaptic dysfunction.Methods:
~500 molecules (including FDA-approved drugs) were profiled using RNA-sequencing in human induced pluripotent stem cell-derived glutamatergic neurons. Drug hits were generated based on their ability to normalize a transcriptomic SYNGAP1 disease profile, and based on their ability to upregulate SYNGAP1 RNA expression, in neurons derived from patients with SYNGAP1 NDD. These hits were further tested in SYNGAP1 patient-derived neurons, using calcium imaging and immunofluorescence. To evaluate in vivo efficacy of nortriptyline, we conducted 24-hour electroencephalography and behavioral assessments, using an established mouse model of Syngap1 haploinsufficiency (+/c.3583-9G >A). Following randomization, nortriptyline was administered to Syngap1+/c.3583-9G >A mice or littermate control mice (Syngap1+/+) at a clinically relevant dose (3 mg/kg) IP daily. Western blot was performed to measure Syngap1 protein levels in brain tissue.Results:
Among profiled small molecules, nortriptyline most effectively reversed disease-associated gene expression patterns in patient-derived neurons, particularly in pathways related to synaptic signaling and glutamatergic activity. Nortriptyline also increased expression of SYNGAP1 RNA, reduced hyperactive network activity, dampened neuronal excitability, increased SYNGAP1 protein levels, and altered its subcellular localization, without affecting the overall number of synapses. Syngap1+/c.3583-9G >A mice exhibited progression of seizure burden between postnatal day 60 (P60) and P120, accompanied by the emergence of pronounced behavioral deficits. Treatment was administered for 30 days beginning at P120, when epilepsy and behavioral deficits were already established. Compared to vehicle-treated controls, nortriptyline-treated Syngap1+/c.3583-9G >A mice showed reduced seizure burden, decreased hyperactivity, improved working memory, and improved social interaction, to levels observed in vehicle-treated wild-type controls. Notably, Syngap1 protein expression was decreased to 64% of vehicle-treated Syngap1+/c.3583-9G >A mice vs. vehicle-treated Syngap1+/+ mice, whereas nortriptyline treatment in Syngap1+/c.3583-9G >A mice restored Syngap1 protein to Syngap1+/+-vehicle levels.
Conclusions:
These preclinical findings indicate that nortriptyline shows promise as a precision therapeutic for SYNGAP1 NDD by correcting SYNGAP1 haploinsufficiency. Our results also indicate that benefit with nortriptyline may be possible when epilepsy and cognitive/behavioral deficits are already present; disease phenotypes may be at least partially reversible. This work underscores the utility of high-throughput drug screening based on transcriptomic profiles, to identify treatments for genetic disorders caused by haploinsufficiency.Funding: Stanford MCHRI, SYNGAP Foundation