Self-regulating gene therapy ameliorates phenotypes and overcomes gene dosage sensitivity in a mouse model of Rett syndrome

Conventional methods of gene transfer lead to inconsistent transgene expression within cells. This variability can be problematic, particularly in conditions like Rett syndrome (RTT), a neurological disorder caused by mutations in the MECP2 (methyl-CpG binding protein 2) gene, because overexpression of MECP2 can also cause adverse effects. To address these challenges, we devised a gene regulation system called Expression Attenuation via Construct Tuning (EXACT), which employs a self-contained, microRNA-based feed-forward loop that not only ensures more consistent transgene expression but also protects against excessive expression. Through cell-based screening assays, we demonstrated the ability of the EXACT circuit to modulate the expression of full-length human MeCP2. Compared to a conventional construct, an EXACT-MECP2 construct exhibited a narrower range of cellular protein expression. Furthermore, the degree of regulation by the EXACT circuit increased with higher transgene doses in vitro and in wild-type mice and mice modeling RTT. Based on cellular and in vivo testing, we identified an optimal configuration for the adeno-associated virus serotype 9 (AAV9) construct for self-regulated MECP2 gene therapy, designated NGN-401. Delivery of NGN-401 to neonatal male Mecp2-/y hemizygous mice via intracerebroventricular injection resulted in prolonged survival and amelioration of RTT-like phenotypes compared to vehicle-treated animals. NGN-401 was also well tolerated by female Mecp2+/- mice and healthy nonhuman primates, in contrast to a conventional construct, which caused toxicity. The results from this study underpin a first-in-human pediatric study of NGN-401 in RTT (ClinicalTrials.gov, NCT05898620). RNA-seq profiling of HEK293A, HepG2, or SH-SY5Y cells transfected with either a plasmid expressing mNeonGreen or one expressing mNeonGreen plus a miRNA (EXACT1).