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 <i>MECP2</i> (methyl-CpG binding protein 2) gene, because overexpression of <i>MECP2</i> can also cause adverse effects. To address these challenges, we devised a gene regulation system called Expression Attenuation via Construct Tuning (EXACT), which uses 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 with a conventional construct, an EXACT-<i>MECP2</i> construct exhibited a narrower range of cellular protein abundance. 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. On the basis of cellular and in vivo testing, we identified an optimal configuration for the adeno-associated virus serotype 9 (AAV9) construct for self-regulated <i>MECP2</i> gene therapy, designated NGN-401. Delivery of NGN-401 to neonatal male <i>Mecp2^-/y</i> hemizygous mice via intracerebroventricular injection resulted in prolonged survival and amelioration of RTT-like phenotypes compared with vehicle-treated animals. NGN-401 was also well tolerated by female <i>Mecp2^+/-</i> mice and healthy juvenile nonhuman primates, in contrast with a conventional construct, which caused toxicity. The results from these studies underpin a first-in-human pediatric trial of NGN-401 in RTT (ClinicalTrials.gov, NCT05898620).