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.

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.