A Self-regulating Gene Therapy for Rett Syndrome

Rett syndrome (RTT) is a neurological disorder caused by loss-of-function mutations in the X-linked MECP2 gene. Genetherapy has emerged as an attractive treatment option for RTT; however, overexpression of MeCP2 protein can lead to adverse effects. Conventional gene therapy vectors can be problematic for dosage sensitive conditions such as RTT, as uneven biodistribution leads to variable levels of transgene expression within cells. To address these challenges, we developed EXACT, a novel transgene regulation system that utilizes a self-contained miRNA-based feed-forward loop. The EXACT system ensures more consistent transgene expression, acting as a safety measure against excessive expression levels. We demonstrated the ability of EXACT circuits to constrain expression of transgenic MeCP2 in vitro and in vivo. When compared to conventional, ‘unregulated’ gene therapy constructs, EXACT-regulated constructs displayed a narrower range of cellular MeCP2 expression. In addition, the degree of regulation increased with higher transgene doses. Following extensive in vivo testing in RTT mouse models, we identified an optimal configuration of the AAV9 construct, designated NGN-401, for self-regulated MECP2 gene therapy. Delivery of NGN-401 to male Mecp2-/yhemizygous mice via intracerebroventricular injection resulted in extended survival and efficacy, ameliorating RTT-like phenotypes. NGN-401 also demonstrated tolerability in female Mecp2+/- heterozygous mice, and in nonhuman primates, in contrast to conventional unregulated constructs, which exhibited toxic effects. These results have supported the advancement of NGN-401 into a first-in-human pediatric study in RTT (ClinicalTrials.gov, NCT05898620).