Familial Dysautonomia (FD) Humanized Disease Models: ELP1 & ELP1 IVS20+6T>C

With the advancement of whole-genome sequencing, it has become increasingly recognized that intronic mutations play a significant role in various diseases, particularly rare diseases. During normal gene transcription, both exons and introns are copied into precursor messenger RNA (pre-mRNA), then the splicing process removes the introns, yielding a coding sequence that is eventually translated into protein. Research indicates that approximately 10%-30% of disease-related gene mutations (mainly intronic mutations) can affect splicing or disrupt regulatory elements like enhancers or silencers, triggering mechanisms like cryptic splice site activation, pseudoexon inclusion, and exon skipping – which ultimately result in disease pathology. These mechanisms often produce premature termination codons (PTCs), triggering nonsense-mediated RNA decay (NMD), alterations in protein secondary structure, or dysregulation of gene/protein expression levels. ^[1-3] Today we discuss humanized gene research models of familial dysautonomia, which is a prime example of a disease caused by a intronic mutations.

Figure 1. Common effects of intronic mutations on pre-mRNA splicing. ^[3]

Familial Dysautonomia (FD) and ELP gene intronic mutations

Familial Dysautonomia (FD) is a rare hereditary neurological disorder caused by impaired neuron development and central nervous system degeneration. Due to defects in the autonomic and sensory nervous systems, patients exhibit symptoms such as excessive sweating, intermittent hypertension, drooling, difficulty swallowing, irregular bowel and bladder control, breathing difficulties, and cyclic vomiting. The disease is associated with certain ethnic and ancestry groups, primarily affecting Ashkenazi Jews, with an incidence rate of approximately 1 in 3,600 in this population. Only about 50% of patients survive to age 40. ^[4-5] 

The ELP1 (IKBKAP) gene encodes a component of the elongator complex, which plays a crucial role in the development and function of neurons. Nearly all FD patients carry biallelic mutations in the ELP1 gene, with over 99% involving a mutation at the 5' splice site of intron 20 (IVS20+6T>C). This mutation disrupts the base pairing between U1 small nuclear ribonucleoprotein (snRNP) and the donor splice site of intron 20, leading to exon 20 skipping. ^[4-6] This mis-splicing causes a frameshift in the transcript reading frame, generating a premature termination codon (PTC), which translates into a truncated ELP protein, ultimately resulting in neuronal damage and cell death.

Figure 2. Mechanism by which the ELP1/IKBKAP intronic mutation IVS20+6T>C leads to abnormal transcript splicing. ^[6]

Familial Dysautonomia: Targeted ELP1 Therapies & Related Animal Models

Currently, there is no cure for FD, and treatment strategies mainly focus on symptomatic relief and supportive care to alleviate symptoms and prevent complications. Since the IVS20+6T>C mutation is the most common pathogenic mutation in FD, research has concentrated on correcting the splicing error caused by this mutation to produce full-length ELP1 protein. Researchers at Cold Spring Harbor Laboratory and PTC Therapeutics have conducted significant studies in this area, including the development of antisense oligonucleotides (ASOs) and small molecule drugs. ^[7-9]

Studies have shown that using wild-type mice (wt mice) to investigate the splicing pattern of intronic mutations in the ELP1 gene is ineffective, as homozygous Elp1 knockout mice die during embryonic development. Transgenic mice expressing the human mutated ELP1 gene do not exhibit clear disease phenotype due to the expression of normal endogenous mouse Elp1 gene levels. This necessitates the combination of Elp1 gene knockdown (single-copy knockout) of the endogenous Elp1 gene in mice with the transgenic gene. However, this approach still faces challenges, such as unstable transgene copies and inconsistent phenotypes. ^[10-12] Additionally, due to differences in splicing patterns between mice and humans, humanizing exon 20 of the mouse Elp1 gene to introduce the IVS20+6T>C mutation along with its flanking introns and has also failed to produce a phenotype. ^[13]

These studies suggest that investigating the splicing pattern of the ELP1 gene in mice may require a longer or even full-length human ELP1 gene sequence.

Figure 3. Regulation of exon 20 splicing pattern of IKBKAP IVS20+6T>C by small molecule drugs. ^[9]

B6-hELP1 Mouse Model: A New Tool for FD Research

To meet the needs for effective FD research, Cyagen has developed a B6-hELP1 humanized mouse model (Product No.: I001203), in which the sequence of the mouse Elp1 gene–from the start codon to the stop codon–is replaced in situ with the corresponding human ELP1 gene sequence. Furthermore, based on this model, we are currently developing  the IVS20+6T>C humanized FD disease point mutation model to support researchers in their FD research.

B6-hELP1 mice successfully express the human ELP1 gene

Expression analysis reveals significant levels of the human ELP1 gene expression in various tissues, including the cortex, kidneys, liver, skeletal muscles, and heart of B6-hELP1 mice, with no detectable expression of the mouse-derived Elp1 gene mRNA.

Figure 4. Expression of the human ELP1 gene and mouse-derived Elp1 gene in B6-hELP1 mice and wild-type mice.

Summary

Current research on FD treatment focuses primarily on correcting the splicing errors caused by mutations to generate full-length ELP1 protein. The B6-hELP1 model (Product No.: I001203) expresses the full-length human ELP1 gene in mice without interference from the endogenous mouse Elp1 gene, making it a valuable tool for FD research. Based on preliminary studies, the B6-hELP1 IVS20+6T>C humanized point mutation model (in development) is expected to exhibit phenotypes similar to those seen in human FD patients.

In addition to the B6-hELP1 models, Cyagen has developed a variety of genetically humanized models for neurological, ophthalmic, and other diseases, providing strong support for researchers developing targeted therapies for various conditions.

Full-Length Genomic Sequence Humanized HUGO Mouse Models

References
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