In vivo characterization of a stochastic, postnatal TSC2-KO mouse model and evaluation of AAV-mediated tuberin replacement therapy
Abstract number :
3.024
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2025
Submission ID :
1157
Source :
www.aesnet.org
Presentation date :
12/8/2025 12:00:00 AM
Published date :
Authors :
Presenting Author: Niyoosha Abdollahpour, BS – Tufts University School of Medicine - Department of Neuroscience
Zin-Juan Klaft, MD, PhD – Tufts University School of Medicine - Department of Neuroscience
Marie Chiara Rehm, MD – Charite Universitaetsmedizin Berlin - Department of Biochemistry
Prahabkar Shilpa, MSc – Massachusetts General Hospital / Harvard Medical School
Edwina Abou-Haidar, MD MB BS – Massachusetts General Hospital / Harvard Medical School
Tomeh Tomeh, MD – Massachusetts General Hospital / Harvard Medical School
Xandra O Breakefield, PhD – Massachusetts General Hospital / Harvard Medical School
Chris Dulla, PhD – Tufts University School of Medicine - Department of Neuroscience
Rationale: Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited neurologic disease caused by loss of function of either tuberin or hamartin resulting in downstream hyperactivation of mTOR. TSC2 patients exhibit tuberin loss and often present with more severe clinical symptoms and TSC2 is understudied in comparison to TSC1. TSC2 patients suffer from high seizure burden that cannot be sufficiently controlled with current anticonvulsants in a majority of patients. Novel treatment approaches are needed to achieve seizure control and AAV-mediated replacement of tuberin has been recently proposed utilizing a condensed construct of tuberin fits within AAV cargo size restrictions. Here, we characterize the survival, seizure burden and distribution of TSC2-KO cells in the stochastic, postnatal, cerebral TSC2-KO mouse model and present preliminary efficacy data of treatment with AAV9 delivering a condensed form of tuberin.
Methods: Intraventricular injections of AAV1-Cre were performed at postnatal day 0 in TSC2fl/fl-Ai9 mice to stochastically induce TSC2-KO. IHC was used in adult mice to evaluate the distribution of TSC2-KO and to assess mTOR activity by quantifying the phosphorylated form of S6 ribosomal protein (pS6 intensity). Patch-clamp recordings from pyramidal neurons in acute slices were performed to assess synaptic input and intrinsic excitability after TSC2-KO and control. In vivo chronic ECoG recordings to assess seizure burden were performed for two weeks starting P42 and analyzed for epileptiform activity. AAV9 were utilized to deliver a condensed form of tuberin systemically via retroorbital injections at P28.
Results: Successful postnatal TSC2-KO was indicated by tdTomato (tom) expression after AAV1-Cre infection across the brain. About 10 % of neurons in somatosensory cortex were TSC2-deficient (tom+) at P35. TSC2-KO cells consistently showed higher pS6 intensities and increased cell size compared to controls confirming downstream mTOR hyperactivation after postnatal TSC2-KO. Electrophysiological assessment of TSC2-KO vs TSC2-competent neurons with patch clamp recordings suggest hypoexcitability of neurons with TSC2-KO. ECoG recordings in adult mice revealed in vivo seizures and sufficiently long survival of stochastic TSC2-KO mice allowing for treatment intervention studies. Preliminary results after P28 systemic application of AAV9-cTub suggest anticonvulsant efficacy as mice treated with AAV9-cTub had lower seizure incidence compared to control.
Conclusions: Postnatal, intracerebroventricular injections of AAV1-Cre in TSC2-floxed mice at P0 leads to mTOR hyperactivity in infected neurons and in vivo seizures during adulthood. Sufficiently long survival of TSC2-KO mice allows for testing of novel treatment approaches. Excitingly, preliminary efficacy data suggests that seizure burden can be reduced in stochastic TSC2-KO mice by AAV9-mediated delivery of condensed tuberin.
Funding: MGB Gene and Cell Therapy Institute (XB)
NINDS (XB)
Basic Mechanisms