As proof of principle, we generated four stable iPSCs lines (three of which stably expressed different shRNAs specifically targeting FOXG1 and one expressed a non-targeting shRNA control) from a proband-derived iPSC line (07-P#9). To confirm stable downregulation of FOXG1 expression, we performed qPCR analyses at TD11 (Figure 6A). Introduction of two FOXG1-targeting shRNAs (shRNA-2 and 3) down-regulated FOXG1 mRNA expression to a level comparable to that of the unaffected family member (Figure 6A, compare bar 6 and 7 with bar 2). Immunostaining for FOXG1 confirmed that shRNA-2 and 3 were able to down-regulate its expression also at the protein level (Figure 6B–F). We next analyzed the expression of GABAergic markers after FOXG1-RNA interference (RNAi) at the transcript and protein level. At TD11, organoids derived from the iPSC lines stably expressing FOXG1 shRNA-2 and shRNA-3 (07-P#9 shRNA-2 and 3) showed downregulation of DLX1, DLX2, and GAD1 transcripts as compared to the same iPSC line expressing the shRNA control (07-P#9 shRNA-C) (Figure 6G–I). Immunostaining and stereological quantification of DLX1-2 and GAD1 positive cells showed that FOXG1 RNAi restored the normal level of GABAergic neuronal differentiation in proband-derived organoids at both TD11 and TD31 (Figure 6K–M).
