Accelerating Rett Syndrome Research with MeCP2 Humanized Mouse Models

Rett Syndrome (RTT) research holds great promise for uncovering insights into rare neurodevelopmental disorders and advancing therapeutic breakthroughs in genetic medicine. RTT is a rare progressive neurodevelopmental disorder with no effective cure at present. Its onset is closely associated with mutations in the methyl-CpG binding protein 2 (MECP2) gene.^[1] In June 2024, the Rett Syndrome Research Trust (RSRT) launched the "Roadmap To Cures" initiative as part of their mission to find a cure for RTT. This program aims to leverage cutting-edge gene and RNA editing technologies to develop curative treatments for RTT, bringing new hope to patients and their families.^[2] 

Humanized mouse models are essential for preclinical research of genetic diseases, as they allow for precise replication of human genetic mechanisms and disease pathology in a controlled environment, enabling researchers to evaluate the efficacy and safety of emerging therapies in a highly relevant context. Our latest MeCP2 humanized mouse model is a powerful research tool to support the ambitious goal of finding a cure for RTT and MECP2-related genetic diseases.

Figure 1. DNA and RNA Editing Therapies are Key Focus Areas in the RTT Roadmap to Cures.^[3]

Role of Gene and RNA Editing in RTT Treatment

The root cause of RTT lies in loss-of-function (LOF) mutations of the MECP2 gene, which leads to neuronal dysfunction, including impaired synaptic connections and neurotransmitter system abnormalities. However, overexpression of MECP2 (such as in MECP2 duplication syndrome) can also cause severe neurological issues. Therefore, the greatest challenge in RTT treatment is precisely regulating MECP2 expression levels. Traditional adeno-associated virus (AAV)-based MECP2 gene replacement therapies face significant limitations in efficacy and safety due to the difficulty of precisely controlling dosage. Gene therapy targeting MECP2 must strike a precise balance, as both loss-of-function (LOF) mutations and overexpression of MECP2 can lead to severe neurological issues.

In contrast, DNA or RNA editing corrects the endogenous MECP2 gene and preserves the natural regulatory mechanisms of the cell for fine-tuning protein production. When the genetic code of the cell is "corrected" through DNA or RNA editing, the natural mechanism for fine-tuning MECP2 protein levels remains intact, ensuring that the "corrected" MECP2 gene is expressed at normal levels.^[4] Unlike CRISPR, which often introduces specific mutations or deletions, this approach focuses on precise endogenous correction, minimizing potential off-target effects and ensuring regulated expression of the MECP2 protein, thereby enhancing therapeutic safety and efficacy. This makes DNA and RNA editing therapies more effective and less risky in terms of adverse side effects compared to AAV-based supplementation therapies in the field of RTT treatment.

Recognizing this, RSRT has prioritized funding for the development of gene and RNA editing approaches within its “MECP2 Editing Alliance” framework

Figure 2. Some of the Collaborators in the Rett Syndrome Research Trust (RSRT) Roadmap to Cures Initiative.^[4]

How MECP2 Humanized Mouse Models Advance RTT Research

Humanized mouse models are essential for RTT research as they allow for precise replication of human genetic mechanisms and disease pathology in a controlled environment, enabling researchers to evaluate the efficacy and safety of emerging therapies in a highly relevant preclinical context. Previously, we introduced the MeCP2 KO mouse model (Product Code: C001582), which is widely used in research on gene supplementation therapies. However, gene and RNA editing therapies require models expressing the full-length human MECP2 gene or RNA for accurate preclinical evaluations and translational outcomes, making mice expressing only the murine Mecp2 gene insufficient for such studies.

To address this, Cyagen has developed the following advanced wild-type and point mutation humanization models for preclinical research of MECP2-related disorders, such as Rett Syndrome:

• B6-hMECP2 Mouse Model (Product ID: C001568): This model replaces the endogenous mouse Mecp2 gene with the full-length human MECP2 gene, including all introns and exons, achieving true humanization.
• B6-hMECP2*T158M Mouse Model (Product ID: C001569): This model introduces the most common RTT-associated missense mutation, T158M,^[5-7] into the humanized MECP2 gene. This model perfectly recapitulates RTT’s pathological features and serves as an ideal platform for preclinical evaluation of DNA and RNA editing therapies.
  
  

Key features of our MECP2 humanized mouse models are shown in the following data.

Figure 3. MECP2 T158M (p.Thr158Met) is the Most Common Pathogenic Missense Mutation in Typical RTT.^[5-7]

Gene Expression Detection

RT-qPCR results show that both homozygous B6-hMECP2 mice and B6-hMECP2*T158M mice significantly express the human MECP2 gene in the brain, with no expression of the murine Mecp2 gene.

Figure 4. Gene Expression Detection in the Brains of Wild-Type Mice (WT), B6-hMECP2 Mice, and B6-hMECP2*T158M Mice.

Survival Curve

Compared to wild-type male mice and B6-hMECP2 male hemizygous mice, the survival rate of hemizygous male B6-hMECP2*T158M mice are significantly reduced. At 24 weeks, only one male hemizygous mouse (hMeCP2^T158M/y) remains alive. The survival rate of female homozygous mice (hMeCP2^T158M/T158M) is approximately 50%, while all other female mice demonstrated 100% survival at 24 weeks.

Figure 5. Comparison of Survival Curves Between Wild-Type Mice, B6-hMECP2 Mice, and B6-hMECP2*T158M Mice.

Brain Pathological Analysis

Brain weight measurements show that the brain weight of B6-hMECP2*T158M mice are significantly lower than those of wild-type mice. Histopathological analysis reveals that in this model, the density of pyramidal cell layers in the hippocampus is increased, the hippocampal area is reduced, and the morphology is abnormal. Silver staining results indicate that the neurons in B6-hMECP2*T158M mice exhibit underdevelopment of neuronal cell bodies (soma) and axons, with a reduced number of axons that are thinner and shorter.

1) Brain weight

2) HE staining & Nissl staining

3) Silver Staining

Figure 6. Comparison of Brain Weight and Histological Staining Results (H&E, Nissl, and Silver Staining) Between Wild-Type Mice and B6-hMECP2*T158M Mice.

Growth and Disease Scoring

The body weight and body length of B6-hMECP2*T158M mice are significantly lower than those of other groups. Mild RTT phenotypes appear at 4 weeks of age, become more pronounced at 6 weeks, and progressively worsening with age.

Figure 7. Body Weight, Body Length, and Disease Phenotype Scoring in Wild-Type Mice, B6-hMECP2 Mice, and B6-hMECP2*T158M Mice.

Morphological and Neurodevelopmental Characteristics

B6-hMECP2*T158M mice exhibit significant cranial abnormalities, including reduced head circumference, shortened muzzle, rough fur, and poor mental state. Around 8–9 weeks of age, approximately 70% of these model mice show hindlimb clasping, a behavior rarely observed in wild-type mice and B6-hMECP2 mice.

Figure 8. Representative Images of Wild-Type Mice, B6-hMECP2 Mice, and B6-hMECP2*T158M Mice.

Behavioral Analysis

Grip strength and rotarod test results indicate that B6-hMECP2*T158M mice exhibit reduced grip strength, impaired motor activity, and coordination. Gait analysis shows that these mice have wider hindlimb stride width and shorter stride length, with pathological changes becoming more pronounced over time.

Figure 9. Grip Strength Test, Rotarod Test, and Gait Analysis Results in Wild-Type Mice, B6-hMECP2 Mice, and B6-hMECP2*T158M Mice.

Applications of Humanized MECP2 Mouse Models

Both our humanized wild-type B6-hMECP2 and humanized point mutation B6-hMECP2*T158M mouse models offer researchers a robust platform for:

• Investigating RTT pathogenesis
• Conducting drug screening and efficacy studies
• Developing and validating gene and RNA editing therapies
• Preclinical evaluation of treatment strategies
  

Conclusion

Our B6-hMECP2 humanized mouse carrying the normal human wild-type MECP2 gene copy (Product Code: C001568) exhibits relatively normal phenotypes and can serve as a baseline model for preclinical therapeutic evaluations. This model serves as a wild-type standard for developing RTT humanized disease models carrying other common pathogenic mutations to quickly serve a broader range of research needs.

The B6-hMECP2*T158M mouse model represents a breakthrough tool for RTT research, offering unparalleled fidelity in replicating human disease pathology. Developed by replacing the mouse endogenous Mecp2 gene with the human MECP2 gene carrying the most common missense mutation T158M found in RTT. This model recapitulates the phenotypic characteristics of RTT and provides a genetically humanized model for effective preclinical research and development of gene editing therapies.

Combined with Cyagen’s advanced CRO services, these models are poised to enable accelerated development of therapeutic strategies - including gene and RNA editing approaches - and bring hope to RTT patients worldwide.

Comprehensive Rodent Neurobehavioral & Preclinical CRO Services

Cyagen provides end-to-end neuroscience research solutions, including:

• Model construction
• Behavioral analysis
• Drug efficacy evaluations
  

Our neurobehavioral testing platforms ensure high-quality data for preclinical studies involving rodent models.
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Extensive Neurodegenerative Disease Models

Cyagen has developed a series of gene-edited mouse models targeting neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Additionally, to meet researchers' needs, customized or collaborative gene-edited mouse models can be developed, including gene knockout, gene knock-in, point mutations, and humanized mouse models, accelerating the progress of neuropharmacological validation experiments.

We also offer customized solutions for gene-edited mouse models, including knockouts, knock-ins, point mutations, and humanizations. For further inquiries about our models or CRO services, you may connect with our team here.

References
[1]Percy AK, Ananth A, Neul JL. Rett Syndrome: The Emerging Landscape of Treatment Strategies. CNS Drugs. 2024 Nov;38(11):851-867. doi: 10.1007/s40263-024-01106-y. Epub 2024 Sep 9. 
[2]Reverse Rett. "RSRT Launches Roadmap to Cures." Last modified October 26, 2024. Accessed December 26, 2024. https://reverserett.org/news/press-releases/rsrt-launches-roadmap-to-cures/.
[3]Reverse Rett. "RSRT Cures." Accessed December 26, 2024. https://reverserett.org/cures/.
[4]Reverse Rett. "Kicking Off RSRT's New MECP2 Editing Consortium." Accessed December 26, 2024. https://reverserett.org/news/articles/kicking-off-rsrts-new-mecp2-editing-consortium/
[5]Gold WA, Percy AK, Neul JL, Cobb SR, Pozzo-Miller L, Issar JK, Ben-Zeev B, Vignoli A, Kaufmann WE. Rett syndrome. Nat Rev Dis Primers. 2024 Nov 7;10(1):84.
[6]Croci S, Carriero ML, Capitani K, Daga S, Donati F, Frullanti E, Lamacchia V, Tita R, Giliberti A, Valentino F, Benetti E, Ciabattini A, Furini S, Lo Rizzo C, Pinto AM, Conticello SG, Renieri A, Meloni I. High rate of HDR in gene editing of p.(Thr158Met) MECP2 mutational hotspot. Eur J Hum Genet. 2020 Sep;28(9):1231-1242.
[7]Ehrhart F, Jacobsen A, Rigau M, Bosio M, Kaliyaperumal R, Laros JFJ, Willighagen EL, Valencia A, Roos M, Capella-Gutierrez S, Curfs LMG, Evelo CT. A catalogue of 863 Rett-syndrome-causing MECP2 mutations and lessons learned from data integration. Sci Data. 2021 Jan 15;8(1):10.