SD-hGFAP Rat

Figure 1. Gene editing strategy of SD-hGFAP Rat. The sequences from the ATG start codon to 3'UTR of the rat Gfap gene will be replaced with the sequences from the ATG start codon to 3'UTR of the human GFAP gene.

● Research on Alexander's Disease (AxD);

● Research on traumatic brain injury.

1. Detection of gene expression

Figure 2. Gene expression in the cerebellum and spinal cord of wild-type (SD) and SD-hGFAP rats. RT-qPCR results revealed significant expression of the human GFAP gene in both the cerebellum and spinal cord of SD-hGFAP rats, while the murine Gfap gene was not expressed. In contrast, SD rats only exhibited expression of the rat Gfap gene, with no expression of the human GFAP gene.

1. Basic information about the GFAP gene

https://rddc.tsinghua-gd.org/gene/2670

2. GFAP clinical variants

3. Disease introduction

Alexander's Disease (AxD), also known as fibrous protein malnutrition or giant brain infantile white matter malnutrition, is a rare non-familial fatal white matter brain disease and progressive neurodegenerative disorder. It is inherited in an autosomal dominant manner. The disease is classified into three types based on the age of onset: infantile, juvenile, and adult, with infantile and juvenile patients being the most common. Infantile AxD is associated with severe intellectual disability, developmental delay, hydrocephalus, and seizures, and patients usually die within 2 years of onset. Common symptoms in juvenile and adult patients include speech abnormalities, dysphagia, seizures, and ataxia. Alexander's disease is caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP).

4. GFAP gene and mutations

The GFAP gene is located on chromosome 17q21 and contains 9 exons. It encodes a neuroglial fibrillary acidic protein that is specific to astrocytes in the central nervous system. This protein is an intermediate filament of astrocytes that participates in intracellular cytoskeletal reorganization, cell adhesion, and maintenance of myelin formation in the brain and neuronal structure and serves as a cell signaling pathway. However, mutations in the GFAP gene lead to protein misaccumulation, resulting in the formation of abnormal inclusions known as Rosenthal fibers. These fibers can cause damage to the brain’s white matter (myelin sheath) in patients with AxD.

Studies have shown that Exon 1 of the GFAP gene may be a hot mutation region for Chinese AxD patients, with the vast majority being point mutations and a few being deletion mutations, while large segment deletions have only been reported once. Four AxD hot mutations have been reported abroad: P.Arg239 (20.3%), P.Arg79 (16.6%), P.Arg88 (7.9%), and P.Arg416 (5.6%). In addition, exon mutations are mainly concentrated in Exon 1 and Exon 4 ^[4-6].

5. Function of non-coding DNA sequences

The c.619-1G>A mutation of the typical splicing acceptor site in Intron 3 can cause Alexander's disease [8]. miRNA targeting the 3’UTR region can regulate GFAP expression ^[9].

6. GFAP-targeted gene therapy

Several GFAP-targeting drugs are currently undergoing clinical or preclinical trials, especially small-molecule drugs and ASO drugs. Their indications include Alexander's disease and traumatic brain injury. Ionis, a leading gene therapy company, has developed an ASO drug called ION-373 (Phase 3 clinical trial) that can inhibit abnormal accumulation of GFAP in rat tissues and restore normal functional expression of astrocytes by targeting GFAP mutant genes. The preclinical study of this pipeline is based on rats with the GFAP R239H mutation. Literature suggests that compared to mouse models, rats with point mutations can exhibit more severe behavioral symptoms, white matter abnormalities, and myelin phospholipid defects, which are more consistent with clinical manifestations in patients ^[10-11]. For this purpose, Cyagen has developed the SD-hGFAP rat model (catalog number: IR1019) and subsequently generated a humanized disease model based on this strain. The disease model carries a common severe early-onset Alexander's Disease (AxD) mutation found in humans (p.R239H).

7. Summary

The GFAP gene is an important pathogenic gene for Alexander's disease and can also serve as a drug target for traumatic brain injury. GFAP whole-genome humanized model and hot mutation models (such as hGFAP (p.R239H) rats) from Cyagen can be used for preclinical research on gene therapy for AxD. Furthermore, Cyagen can provide customized services for different point mutations of the GFAP gene.