B6-hMECP2 Mice

Figure 1. Gene editing strategy of B6-hMECP2 mice. The mouse MECP2 protein coding sequence for amino acids 10–484 was replaced with the corresponding human MECP2 protein coding sequence for amino acids 10–486 because the coding sequences for amino acids 1–9 of mouse and human MECP2 proteins are identical.

● Research on Rett syndrome (RTT).

1. Detection of human MECP2 gene expression

Figure 2. Human MECP2 gene expression in the lungs and brains of 6-week-old female wild-type (B6N) and B6-hMECP2 mice. RT-qPCR analysis showed that the human MECP2 gene was expressed in both the lungs and brains of B6-hMECP2 mice, but not in wild-type mice.
ND: Not detected

 

2. Detection of mouse Mecp2 gene expression

Figure 3. Mouse Mecp2 gene expression in the lungs and brains of 6-week-old female wild-type (B6N) and B6-hMECP2 mice. RT-qPCR analysis showed that the mouse Mecp2 gene was expressed in both the lungs and brains of wild-type mice, but not in B6-hMECP2 mice.

1. Basic information about the MECP2 gene

https://rddc.tsinghua-gd.org/en/gene/4204

 

2. MECP2 clinical variants

3. Disease introduction

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder that occurs predominantly in female infants and young children, with an incidence of approximately 1/10,000 to 1/15,000 females. Clinical features include intellectual disability, loss of language function, stereotyped hand movements, and gait abnormalities. Affected children typically have normal development in the early stages, followed by stagnation of head circumference growth at 6–18 months of age and regression of acquired skills. Overt cognitive and motor impairments develop 1–2 years later. Mutations in the methyl-CpG binding protein 2 (MECP2) gene account for >90% of RTT cases. Normally, females have one functional MECP2 copy on each X chromosome; however, in most cases of RTT, patients have only one mutated MECP2 copy among their two copies. This is because X chromosome inactivation in neurons silences the other normal MECP2 copy, resulting in insufficient MECP2 protein expression and RTT ^[1].

4. MECP2 gene and mutations

The MECP2 gene is located on the X chromosome and encodes a nuclear protein that binds to methylated DNA to regulate gene transcription and expression. Repetitive mutations in MECP2 can cause MECP2 duplication syndrome (MDS), while functional deficiency of MECP2 can impair production of this nuclear protein, leading to central nervous system functional maturity disorders that affect learning and memory functions and cause Rett syndrome (RTT). Over 100 MECP2 gene mutations have been identified to date, with more than 80% being cytosine-to-thymine (C>T) transitions. Hotspot mutations, which account for 70% of all mutations, include R106W, R133C, T158M, R168X, R255X, R270X, R294X, and R306C. T158M is the most common mutation type.

5. Function of non-coding DNA sequences

Research indicates that pathogenic mutations exist in the introns of the MECP2 gene ^[3].

6. MECP2-targeted gene therapy

RTT treatment mainly focuses on MECP2 gene supplementation therapy based on AAV vectors. This involves delivering human MECP2 genes through AAV vectors to compensate for the deficiency of MECP2 genes in patients. However, the large size of the MECP2 gene exceeds the delivery capacity of most vectors, and overexpression of the MECP2 gene can also lead to serious neurological diseases, limiting the development of this therapy. Therefore, gene/RNA editing to repair MECP2 gene mutations and restore normal expression of MECP2 protein has received widespread attention. Currently, multiple research groups have used CRISPR-based gene editing technology to repair mutations in the MECP2 gene in induced pluripotent stem cells (iPSCs) or ex vivo patient cells [1,2]. Additionally, some studies have used transgenic humanized mice for in vivo pharmacological evaluations. For example, researchers have shown that human-specific MECP2-ASO drugs can effectively downregulate the overexpression of MECP2 in the brains of transgenic humanized MDS mice (Mecp2-/Y; MECP2-TG1; MECP2-GFP). The drug can alleviate various behavioral defects caused by excessive expression of MECP2 and restore normal expression in a dose-dependent manner [4]. However, transgenic humanized mice used in such in vivo experiments have defects, such as complex construction, insufficient copy number, random insertion, and insufficient humanization region. More efficient in vivo gene editing models are yet to be developed.

7. Summary

Rett syndrome (RTT) is a severe neurological disorder that arises from mutations in the MECP2 gene. Humanized MECP2 mice serve as an invaluable resource for conducting preclinical research on RTT and for the development of gene therapy drugs. Cyagen provides whole-genome humanized MECP2 mouse models, which carry the entire human MECP2 gene and can produce humanized MECP2 mice with specific point mutations to facilitate the study of RTT pathogenesis and the development of therapies targeting MECP2.