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E - Disease Models and Clinical Applications -> Neurologic Diseases

834: A Self-regulating Gene Therapy for Rett Syndrome

Type: Oral Abstract Session

Presentation Details
Session Title: Novel Therapeutic Targets to treat CNS Disorders
Location: Room 202
Start Time: 5/18/2022 16:00
End Time: 5/18/2022 16:15

Introduction: Rett syndrome (RTT) is a neurological disorder caused by mutations in the X-linked MECP2 gene. Mecp2-null mice recapitulate the cardinal features of the disorder and gene reactivation studies using conditional alleles lead to robust phenotypic correction. Whilst this makes RTT an attractive gene therapy target, MECP2 is a dosage sensitive gene, with both animal studies and the human duplication disorder suggesting that MeCP2 levels need to be kept within a narrow range to achieve efficacy while avoiding overexpression related toxicity. These challenges are magnified by the variable biodistribution pattern of commonly used AAV vectors, which lead to hotspots of expression as well as excessive transgene expression in sensitive cell types. Approach: To overcome these challenges, we have developed a single gene circuit in which transgene expression is regulated by a miRNA-based feedforward loop (termed EXACT). This circuit provides a cell autonomous mechanism to prevent overexpression in strongly transduced cells whilst still allowing expression of therapeutic protein levels in more modestly transduced targets. Importantly, the miRNA sequence is not based on any existing mammalian miRNA, thus preventing interference with endogenous miRNA-mRNA gene regulation in transduced cells. In-vitro testing: In order to test the ability of the EXACT circuit to regulate protein levels and tune the setpoint of expression, we designed a cell-based fluorescent screening assay in which candidate MECP2 transgenes were fused to a reporter, whilst a second reporter in a separate transcriptional unit acted as a surrogate for gene dose. For unregulated constructs, MeCP2 levels increased proportionally with plasmid dose, whilst for regulated constructs, protein levels displayed relative dosage insensitivity and were maintained within a much narrower range or setpoint. Efficacy and Safety: To demonstrate efficacy in vivo, we delivered either regulated or unregulated MECP2 constructs to male Mecp2 knockout mice using AAV9. This severe mouse model displays reduced lifespan (median survival = 11.7 weeks) and prominent respiratory and motor impairments. Mice dosed by neonatal intracereboventricular (ICV) injection with 1e11 vg/mouse of the lead regulated MECP2 construct showed significant improvement in survival (median survival = 22.9 weeks) and a concomitant improvement in RTT-like clinical score. In contrast, mice dosed with the unregulated constructs did not show any improvement in survival, possibly due to overexpression toxicity. At a higher dose of 3e11 vg/mouse, mice treated with the regulated lead construct showed a profound improvement in lifespan (median survival > 35 weeks) and significant amelioration of RTT-like phenotypes. At this higher dose, mice treated with the unregulated construct showed severe signs of MeCP2 overexpression and most were euthanised at ~3 weeks. These data demonstrate the ability of the EXACT circuit to enable strong efficacy and significantly improve the safety profile of an MECP2 gene therapy vector. In separate toxicity studies, early in-life safety was demonstrated in non-human primates treated with the lead construct. Conclusion: Overall, these data support the use of the EXACT technology in the further development of an effective gene therapy for Rett syndrome that avoids the safety concerns with conventional unregulated gene replacement.

Stuart Cobb1,2, Paul Ross2, Ralph D. Hector2, Kamal K. Gadalla2, Sophie Thomson2, Jim Selfridge2, Nicholas W. Keiser1, Jennifer L. Daily1

1Neurogene Inc., New York, NY,2Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
  S. Cobb: 1; Commercial Interest i.e. Company X; Neurogene. 1; What was received? i.e. Honorarium; Consulting fee, Stock options. 1; For what role? i.e. Speaker; Consulting.

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