USH2A Humanized Disease Model for Usher Syndrome Type II Research

Usher Syndrome (USH) is a genetic disorder that causes both hearing and vision loss due to mutations in genes which play a critical role in inner ear and retinal function. The most common subtype is Usher syndrome type II (USH2). USH2A gene mutations lead to Usher syndrome type IIA (USH2A), which represents over half of all USH2 cases and is closely associated with retinitis pigmentosa, a progressive degenerative eye disease.

Current research on USH treatments primarily focuses on exon skipping and gene editing technologies, including emerging approaches like CRISPR and antisense oligonucleotides (ASO).

To address the urgent need for effective therapies, Cyagen has developed advanced USH2A humanized mice that offer a precise platform for preclinical research. These humanized disease models are designed to accelerate the development of exon-skipping and gene-editing therapies, making them an invaluable tool in the pursuit of treatments for Usher Syndrome.

What is Usher Syndrome (USH)?

Usher syndrome (USH), also known as hereditary deafness-retinitis pigmentosa syndrome, has an incidence of approximately 1 in 5,000 to 1 in 16,000. All forms of USH are inherited in an autosomal recessive manner, with types I and II being the most common. Patients with USH typically present with mild to moderate hearing loss and develop symptoms of RP in adulthood.^[1-2] The disease is likely underdiagnosed due to the progression of the disease and diagnostic challenges, so the actual incidence of USH may be higher than current estimates. Current research on USH treatments primarily focuses on exon skipping and gene editing technologies, including emerging approaches like antisense oligonucleotides (ASO) and CRISPR.^[2] More than 400 different mutations in the USH2A gene have been identified in individuals with Usher syndrome type II (USH2), accounting for more than half of all cases of USH2.

Cyagen has developed a humanized USH2A (hUSH2A) mouse model designed for research and evaluation of therapies implementing exon skipping and gene editing targeting USH2A.

Figure 1. USH2A is the most common pathogenic gene mutation in Usher syndrome (USH). ^[2]

Why Target USH2A for Usher Syndrome Research?

Usher syndrome type II (USH2) is the most common subtype of USH, with around 57% to 79% of USH2 patients carrying mutations in the USH2A gene, which encodes the Usherin protein. This protein is essential for the development and maintenance of homeostasis in the inner ear and retina.^[2-3] Usherin contains laminin EGF-like motifs, a pentapeptide domain, and several fibronectin type III motifs, and it is primarily expressed in the basement membrane. It plays a crucial role in the development of inner ear hair cells and auditory signal transduction, while in the retinal basement membrane, it regulates adhesion functions through its interactions with fibronectin.^[2-3] Mutations in the USH2A gene lead to truncated or domain-deleted Usherin proteins and disrupt normal protein function, interfering with normal inner ear hair cell development and causing abnormal fibronectin assembly in the retinal basement membrane. This ultimately results in hearing loss and symptoms of retinitis pigmentosa (RP).

Figure 2. Schematic representation of Usherin protein encoded by USH2A, showing its localization and function in photoreceptor and hair cells.^[2]

Current Research on USH2A Targeted Therapies

Although no therapies for USH2 have been approved yet, the primary focus of ongoing research is targeting the USH2A gene. One major challenge is the large size of the USH2A gene (over 800k base pairs) and its coding sequence (CDS) (exceeding 15k base pairs), which exceeds the carrying capacity of traditional adeno-associated viral (AAV) vectors. This has led researchers to focus on targeting exon 13 of the USH2A gene, a hotspot region for pathogenic mutations associated with USH2. Two common mutations in this region, c.2299delG and c.2276G>T, are key targets for therapies like ASO-mediated exon skipping and CRISPR-based gene editing.^{[2, 4-6]}

Multiple studies are currently focused on this region, including therapies based on ASO and gene editing, aiming to restore normal Usherin protein expression either by repairing the mutation through gene editing or by exon skipping mediated by ASO.^[7-9] 

Given the genetic differences between humans and mice, and the fact that both ASO and gene editing therapies target the human USH2A gene, Cyagen has developed the B6-hUSH2A(E10-15) humanized mouse model (Product No.: C001554). This model includes human USH2A exon 13, making it ideal for effectively evaluating and advancing these therapies toward clinical trials.

Figure 3. Gene editing therapy and ASO-mediated exon skipping are the primary research directions for USH treatment. ^[9]

B6-hUSH2A(E10-15) Humanized Mouse Model Design

The start codon (ATG) of the mouse Ush2a gene is located in exon 1, whereas in the human USH2A gene, it is located in exon 2. Therefore, exon 13 of the human USH2A gene corresponds to exon 12 of the mouse Ush2a gene. The B6-hUSH2A(E10-15) humanized mouse model (Product No.: C001554) was developed by replacing exons 9 to 14 of the mouse Ush2a gene and their flanking sequences with exons 10 to 15 of the human USH2A gene [hUSH2A(E10-15)] and their flanking sequences. This model ensures precise expression of exon 13 from the human USH2A gene, with humanized sequences flanking it. Gene sequencing results confirm that the Ush2a gene in the model has been successfully replaced by the corresponding sequences of the human USH2A gene. Its base sequence aligns with the reference sequence of the human USH2A gene, indicating that the model is suitable for further studies on ASO and CRISPR-based therapies.

Figure 4. Humanization strategy and regional sequencing results of the B6-hUSH2A(E10-15) mouse model.

Phenotypic Characteristics of B6-hUSH2A(E10-15) Mice

Since the human USH2A gene in the B6-hUSH2A(E10-15) mouse model is expressed in its wild-type form, these mice exhibit the same healthy phenotype as wild-type mice, including normal retinal morphology, retinal vasculature, and photoreceptor function.

Figure 5. Fundus morphology, retinal optical coherence tomography (OCT), fundus fluorescein angiography (FFA), and electroretinography (ERG) results of wild-type (WT) mice and B6-hUSH2A(E10-15) mice.

Applications of the B6-hUSH2A(E10-15) Humanized Mouse Model

• Exon Skipping Research: This model supports studies on ASO-based therapies targeting exon 13 of the USH2A gene, a key area for mutation in USH2 patients.
• CRISPR Gene Editing: As CRISPR gene-editing therapies gain momentum, this model provides an essential tool for testing how effectively gene editing can restore normal Usherin protein production.
• Therapeutic Development: The humanized model’s accuracy ensures that therapies designed for USH2A mutations in humans can be evaluated and optimized before clinical trials.

Conclusion: Advancing Usher Syndrome Research with Cyagen’s Humanized Models

Research and development of USH2A-targeted therapies is primarily focused on exon 13 and its surrounding regions, utilizing technologies such as ASO and CRISPR gene editing. The B6-hUSH2A(E10-15) humanized mouse model (Product No.: C001554) successfully expresses the human USH2A gene while maintaining normal retinal morphology and function. Given that the base sequence fully matches the reference sequence of the human USH2A gene, the B6-hUSH2A(E10-15) humanized mouse model serves as an essential platform for studying therapies targeting the human USH2A gene, including ASO and CRISPR gene editing. Additionally, Cyagen has developed a humanized disease model based on this platform that carries the most common USH2 pathogenic mutation (c.2299delG), simulating human disease phenotypes and meeting the needs for precision therapy development. Interested researchers are welcome to inquire for more information.

Cyagen develops a variety of genetic disease models, inducible disease models, and humanized models in the field of retinal diseases, while also offering comprehensive ophthalmology CRO services. For more information about Cyagen’s USH2A humanized disease models or to inquire about our comprehensive ophthalmology CRO services, please contact us.

Ophthalmic Disease Models

Cyagen’s Next-Generation Humanized Mouse Models: HUGO

Cyagen has launched the HUGO (Humanized Genomic Ortholog) Project, inviting global partners to collaborate on developing novel fully humanized models to support new drug development.

HUGO-GT™ Next-Generation Humanized Models

The Humanized Genomic Ortholog for Gene Therapy (HUGO-GT™) mouse model offers a higher degree of humanization compared to traditional models to serve as an effective evaluation platform, especially for gene therapy drugs with high requirements for gene sequence integrity, such as ASO, CRISPR, and siRNA.

Our HUGO-GT^TM fully humanized genome mice are developed based on the proprietary TurboKnockout-Pro technology to achieve in situ replacement of mouse genes, encompassing a broader range of intervention targets and providing full coverage of pathogenic gene mutation sites-all without patent or ownership disputes. The fully humanized target genes within these models are consistent with the pathogenic genes carried by humans and cover the majority of drug targets, significantly enhancing screening efficiency for various types of preclinical drug experiments.

HUGO-Ab™ Mice for Fully Human Antibody Discovery

HUGO-Ab^TM (HUmanized Genomic Ortholog for Antibody Development) mice represent a leap forward in antibody discovery models:  with fully humanized genes in the antibody variable regions, these mice are capable of producing fully humanized antibodies in vivo with high affinity and low immunogenicity. Our TurboKnockout® ES technology replaces the VH and VL genes in situ to offer a higher degree of humanization with more stable phenotypic and functional outcomes in progeny than traditional transgenic methods.

HUGO-Ab™ Mouse Models for Antibody Discovery

* Fully human, full sequence diversity (Heavy, kappa, Lambda)

* Robust immune response for efficient discovery

* Human-like immune profile for vaccine development

* Numerous fixed light chain models

* Single domain models for monobody discovery

* Multiple backgrounds (B6, Balb/c, SJL)

High-throughput Fully Humanized Antibody Discovery Platform

Combining Biointron’s AbDrop™ with Cyagen’s HUGO-Ab™ mice streamlines the discovery of fully human antibodies to as few as 3 months. Our innovative High-throughput Fully Humanized Antibody Discovery Platform simplifies and accelerates drug development by eliminating the need for complex genetic modifications,  reducing costs, and leading to safer, more effective antibody therapies.

References
[1]Delmaghani S, El-Amraoui A. The genetic and phenotypic landscapes of Usher syndrome: from disease mechanisms to a new classification. Hum Genet. 2022 Apr;141(3-4):709-735. 
[2]Toualbi L, Toms M, Moosajee M. USH2A-retinopathy: From genetics to therapeutics. Exp Eye Res. 2020 Dec;201:108330.
[3]McGee TL, Seyedahmadi BJ, Sweeney MO, Dryja TP, Berson EL. Novel mutations in the long isoform of the USH2A gene in patients with Usher syndrome type II or non-syndromic retinitis pigmentosa. J Med Genet. 2010 Jul;47(7):499-506.
[4]Pendse ND, Lamas V, Pawlyk BS, Maeder ML, Chen ZY, Pierce EA, Liu Q. In Vivo Assessment of Potential Therapeutic Approaches for USH2A-Associated Diseases. Adv Exp Med Biol. 2019;1185:91-96.
[5]Yan D, Ouyang X, Patterson DM, Du LL, Jacobson SG, Liu XZ. Mutation analysis in the long isoform of USH2A in American patients with Usher Syndrome type II. J Hum Genet. 2009 Dec;54(12):732-8.
[6]Dreyer B, Tranebjaerg L, Brox V, Rosenberg T, Möller C, Beneyto M, Weston MD, Kimberling WJ, Cremers CW, Liu XZ, Nilssen O. A common ancestral origin of the frequent and widespread 2299delG USH2A mutation. Am J Hum Genet. 2001 Jul;69(1):228-34.
[7]Dulla K, Slijkerman R, van Diepen HC, Albert S, Dona M, Beumer W, Turunen JJ, Chan HL, Schulkens IA, Vorthoren L, den Besten C, Buil L, Schmidt I, Miao J, Venselaar H, Zang J, Neuhauss SCF, Peters T, Broekman S, Pennings R, Kremer H, Platenburg G, Adamson P, de Vrieze E, van Wijk E. Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations. Mol Ther. 2021 Aug 4;29(8):2441-2455.
[8]Stemerdink M, García-Bohórquez B, Schellens R, Garcia-Garcia G, Van Wijk E, Millan JM. Genetics, pathogenesis and therapeutic developments for Usher syndrome type 2. Hum Genet. 2022 Apr;141(3-4):737-758.
[9]French LS, Mellough CB, Chen FK, Carvalho LS. A Review of Gene, Drug and Cell-Based Therapies for Usher Syndrome. Front Cell Neurosci. 2020 Jul 9;14:183.