Recupero fenotipico del deficit di AGC1 in cellule progenitrici neuronali derivate da hiPSC di pazienti attraverso l'integrazione mirata di AGC1 cDNA wild-type nel locus safe harbor AAVS1 sul cromosoma 19

AGC1 deficiency is a rare and severe early-onset encephalopathy caused by mutations in the SLC25A12 gene. This transporter is a fundamental component of the malate-aspartate shuttle (MAS), and its dysfunction severely impairs the transfer of cytosolic reducing equivalents into the mitochondria, driving the brain into a pathological state of metabolic stress, restricted pyruvate oxidation, and hypomyelination. This study investigates the therapeutic potential and temporal constraints of a targeted gene insertion strategy in patient-derived in vitro models. Using CRISPR/Cas9 technology, a wild-type AGC1 expression cassette, under the control of a doxycycline-inducible promoter, was integrated into the AAVS1 genomic safe harbor of human induced pluripotent stem cells (hiPSCs) derived from two distinct patients: P1, carrying a severe missense mutation, and P2, carrying a compound heterozygous mutation. The engineered hiPSCs were subsequently differentiated into neural progenitors (NPs) under strictly modulated transgene induction time windows. In-depth phenotypic and bioenergetic analyses revealed divergent outcomes depending on the model. In the P1 model, the exogenous restoration of AGC1 failed to rescue the pathological phenotype due to a persistent secondary downregulation of the mitochondrial pyruvate carriers (MPC1/2). Conversely, the P2 model exhibited a complete and striking biological recovery, strictly dependent on the timing of AGC1 induction during the differentiation process. Continuous transgene activation from the onset of neural differentiation fully normalized cell morphology, abolished pathological cell death, and restored glutamine-independent proliferation. Crucially, early AGC1 induction in the P2 model rewired the entire metabolic network: it restored the transcription of MPC1/2 and OGDH, normalized extracellular lactate secretion, and completely rescued mitochondrial Complex I activity. Real-time bioenergetic analysis via Seahorse XF confirmed the complete normalization of the glycolytic rate, the restoration of maximal mitochondrial respiratory capacity, and the re-establishment of a physiological balance between glycolytic and mitochondrial ATP production. In contrast, activating AGC1 expression exclusively after the completion of differentiation yielded no functional recovery. Collectively, these findings demonstrate that timely AGC1 restoration, initiated at the earliest stages of differentiation, is both necessary and sufficient to reverse the metabolic collapse, providing a robust proof-of-concept for early targeted gene therapy interventions in AGC1 deficiency.