Advancing Hamartin Therapy in a TSC1 Mouse Model: AAV Delivery and Enhancement with Extracellular Vesicles
Edwina Abou Haidar1, Tomeh Tomeh1, shilpa Prabhakar1, Roberta Beauchamp1, Koen Breyne1, Ivan Coto Hernandez2, Vijaya Ramesh1, Xandra Breakefield1
1Neurology, 2Radiology, Massachusetts General Hospital
Objective:
To evaluate therapeutic effects of AAV-mediated hamartin delivery in a TSC1 mouse model and investigate the potential of extracellular vesicles (EVs) to enhance hamartin distribution and function, aiming to improve treatment efficacy for TSC1-associated pathologies.
Background:
Tuberous sclerosis complex (TSC) is a dominantly inherited disorder caused by mutations in TSC1 or TSC2, encoding hamartin or tuberin, which bind to form a complex inhibiting mTORC1 signaling. Loss of either protein increases cell growth and proliferation via mTORC1 activation. Neurologic symptoms include refractory seizures, hydrocephalus, and cognitive impairment.
Design/Methods:
In the stochastic, cranial TSC1 mouse model, hamartin loss of function was induced at birth via injection of an AAV vector encoding Cre-recombinase into cerebral ventricles. Therapeutic effects were then assessed with an intravenous AAV9-hamartin vector administered on postnatal-day 21 (D21).
To enhance gene delivery, we explored EVs as vehicles by generating five plasmid constructs to potentially improve hamartin uptake that were transfected into HEK293T cells and assessed by immunoblotting to compare protein expression in both cells and EVs. Transfer of modified hamartin between cells was evaluated using EVs and a membrane permeable to particles < 1 µm.
Results:
Cre-recombinase injection at birth in the TSC1 model caused subventricular cell proliferation, reduced ventricular volume, neuronal enlargement, increased S6 phosphorylation, dysmyelination, and early mortality. Administering AAV9-hamartin on D21 significantly normalized these effects, extending lifespan from 50 to over 120 days in 85% of treated animals that was dose-dependent.
In vitro, the hamartin-V5 construct with an N-terminal palmitoylation signal showed the highest EV incorporation and were successfully taken up by other cells, indicating that the palmitoylation signal enhances hamartin’s EV loading capacity and transfer potential.
Conclusions:
AAV9-mediated hamartin delivery extended lifespan and improved pathology in the TSC1 model, while the palmitoylated variant showed improved EV incorporation, indicating that an AAV-EV approach could enhance therapeutic efficacy for TSC1.
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