The Efficacy of a Human-Ready mini MECP2 Gene Therapy in a Pre-Clinical Model of Rett Syndrome

Abstract

Inactivating mutations and the duplication of methyl-CpG binding protein 2 (MeCP2), respectively, mediate Rett syndrome (RTT) and MECP2 duplication syndrome. These disorders underscore the conceptual dose-dependent risk posed by MECP2 gene therapy for mosaic RTT patients. Recently, a miRNA-Responsive Autoregulatory Element (miRARE) mitigated the dose-dependent toxicity posed by self-complementary adeno-associated viral vector serotype 9 (AAV9) miniMECP2 gene therapy (scAAV9/miniMECP2-myc) in mice. Here, we report an efficacy assessment for the human-ready version of this regulated gene therapy (TSHA-102) in male Mecp2^-/y knockout (KO) mice after intracerebroventricular (ICV) administration at postnatal day 2 (P2) and after intrathecal (IT) administration at P7, P14 (±immunosuppression), and P28 (±immunosuppression). We also report qPCR studies on KO mice treated at P7-P35; protein analyses in KO mice treated at P38; and a survival safety study in female adult Mecp2^-/+ mice. In KO mice, TSHA-102 improved respiration, weight, and survival across multiple doses and treatment ages. TSHA-102 significantly improved the front average stance and swing times relative to the front average stride time after P14 administration of the highest dose for that treatment age. Viral genomic DNA and miniMECP2 mRNA were present in the CNS. MiniMeCP2 protein expression was higher in the KO spinal cord compared to the brain. In female mice, TSHA-102 permitted survivals that were similar to those of vehicle-treated controls. In all, these pivotal data helped to support the regulatory approval to initiate a clinical trial for TSHA-102 in RTT patients (clinical trial identifier number NCT05606614).

Keywords: Rett syndrome (RTT); adeno-associated virus (AAV); methyl-CpG binding protein 2 (MeCP2).

Figure 1

After P2 ICV administration, TSHA-102 is effective in KO mice. (A) TSHA-102 improves KO body weight. Statistical analyses were conducted on KO groups through 11 weeks of age and on WT groups up through 34 weeks of age. (B) TSHA-102 significantly extends KO survival. TSHA-102-treated KO survival is not significantly different from vehicle-treated WT survival. (C) TSHA-102 is well-tolerated in WT mice, with a significant difference in behavior noted at only one time point. (D) TSHA-102 improves post-hoc aggregate severity scores in KO mice at the indicated time points. TSHA-102 delays the onset age of (E) severe hindlimb clasping and (F) severely abnormal gait among KO mice achieving these phenotypes. (A–F) Color-coding in the figure legend for A applies to (A–F). For example, all red lines and bars describe vehicle-treated KO mice. Levels of statistical significance for this and other figures are defined in the Materials and Methods. Throughout this manuscript, vehicle administration is implied in legends where a vector dose is not specified. (A,C–F) Data are mean ± SEM. n per group: (A) 6–14; (B) 13–14; (C) 5–12; (D) 11–12; and (E,F) 10–12. * p ≤ 0.05; ** p ≤ 0.01; and **** p ≤ 0.0001.

Figure 2

TSHA-102 improves weight in older KO mice. (A) After P7 and (B) P14 administration, all doses increase KO weight during the indicated timespans. (C) After P14 and in the presence of immunosuppression, 4.4 × 10¹¹ vg/mouse improves KO weight during the indicated timespan. (D) After P28 administration, each dose increases KO weight during the indicated time points. (E) After P28 administration and in the presence of immunosuppression, each dose increases KO weight during the indicated time points. (A–E) Asterisks for virus-treated KO mice are color-matched to their respective group and indicate statistical comparisons against vehicle-treated KO mice (A,B,D). Red asterisks indicate comparisons between vehicle-treated KO mice and vehicle-treated WT mice. (C,E) A statistical comparison for WT versus immunosuppressed KO mice is not provided because the groups differ by two variables. (A–E). The same WT group is shown across graphs. Data are mean ± SEM. n per group: (A,B) 10–12; (C) 7–12; and (D,E) 12. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; and **** p ≤ 0.0001.

Figure 3

TSHA-102 improves breathing frequency in older KO mice. Although not notated above for simplicity, there was a significant difference between WT and KO vehicle-treated controls for respiratory readouts (p ≤ 0.05). After (A) P7, (B) P14, and (C) P28 administration, the indicated doses of TSHA-102 increased breathing frequency. After (D) P7, (E) P14, and (F) P28 administration, the indicated doses of TSHA-102 decreased expiratory time. (G) TSHA-102 did not improve inspiratory time after P7 administration. After (H) P14 and (I) P28 administration, the indicated doses of TSHA-102 decreased inspiratory time. Data are mean ± SEM. n per group: (A,D,G) 7–10; (B,E,H) 9–12; and (C,F,I) 8–12. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; and **** p ≤ 0.0001.

Figure 4

TSHA-102 improves apnea phenotypes in older KO mice. Although not notated above for simplicity, there was a significant difference between WT and KO vehicle-treated controls for respiratory readouts (p ≤ 0.05). (A) After P7 administration, TSHA-102 did not affect apnea duration. After P14 (B) and P28 (C) administration, the indicated doses of TSHA-102 decreased apnea duration. After P7 (D) and P14 (E) administration, each dose of TSHA-102 decreased apnea frequency. (F) After P28 administration, the indicated doses of TSHA-102 decreased apnea frequency. The lowest dose was not effective at treating apnea phenotypes after P28 administration. Data are mean ± SEM. n per group: (A,D) 7–10; (B,E) 9–12; and (C,F) 8–12. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; and **** p ≤ 0.0001.

Figure 5

TSHA-102 delays the onset of generalized RTT-like phenotypes in KO mice in a subset of treatment paradigms. The approximate onset ages for severe (A–C) breathing, (D–F) clasping, and (G–I) gait were quantified. (A–C) Few vehicle-treated KO mice achieved level 2 scores for abnormal breathing. (B) Virus-treated mice experienced a 48% delayed onset of severely abnormal breathing after P14 administration (p ≤ 0.05). (C) No mice treated with the highest dose at P28 developed severe breathing scores (see broken X axis). (E) After treatment at P14, virus-treated mice demonstrated an approximately 30% delay in the onset age for severe clasping (8.8 × 10¹⁰ vg/mouse; compared to the approximately 200% delay after dose-matched neonatal ICV administration). (I) After P28 administration, mice treated with the highest dose experienced an approximately 70% delay in developing severely abnormal gait. Data are mean ± SEM. n developing severe scores per group: (A) 4–7; (B) 3–6; (C) 2–5; (D) 5–9; (E) 6–10; (F) 7–11; (G) 5–9; (H) 2–11; and (I) 4–7. * p ≤ 0.05; ** p ≤ 0.01.

Figure 6

TSHA-102 significantly changes gait parameters in KO mice after P14 administration. For simplicity, significant differences are notated for comparisons between KO groups only. The same dose (4.4 × 10¹¹ vg/mouse) significantly changed 6 abnormal gait phenotypes: (A) front average stance time, (B) front average swing time/front average stride time, (C) front average stance time/front average stride time, (D) front average stride time, (E) front average normalized stride frequency, and (F) rear average propulsion time. Data are mean ± SEM. n per group is 4–12. *** p ≤ 0.001; and **** p ≤ 0.0001.