B6-hUSH2A(E10-15) Mice

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Catalog Number: C001554

Strain Name: C57BL/6JCya-Ush2atm1(hUSH2A Exon 10-15)/Cya

Genetic Background: C57BL/6JCya

Reproduction: Homozygote x Homozygote

This strain is the HUGO-GT (Humanized Genomic Ortholog for Gene Therapy) mice from Cyagen.

 

Strain Description

Usher syndrome (USH), also referred to as hereditary deafness-retinitis pigmentosa syndrome or retinitis pigmentosa-neurosensory deafness syndrome, is an autosomal recessive disorder marked by genetic heterogeneity. The primary clinical manifestations include congenital sensorineural hearing loss, progressive retinitis pigmentosa (RP), and visual impairment. USH is the leading disorder resulting in deafness and blindness, with an estimated prevalence ranging from 1 in 5,000 to 1 in 16,000 individuals. USH is classified into three subtypes—USH1, USH2, and USH3—based on the age of onset and effects on hearing and vestibular function. Patients with USH1 present with profound congenital deafness and vestibular dysfunction, typically developing RP before adulthood. USH2 is characterized by moderate to severe hearing loss without vestibular dysfunction, with RP symptoms manifesting later in adulthood. USH3 patients are born with normal hearing, which progressively declines alongside the onset of RP. USH1 is the most severe form, while USH2 is the most prevalent, accounting for 40%–50% of cases. However, underdiagnosis and the gradual progression of the disease suggest that the true prevalence of USH2 may be underestimated. The USH2A gene is the primary causative gene for USH2, with 75%–90% of USH2 cases linked to mutations in this gene [1].

The USH2A gene encodes Usherin, a protein featuring laminin EGF-like, pentraxin, and fibronectin type III domains, predominantly expressed in the basement membrane of the inner ear and retina. Usherin plays a critical role in developing hair cells in the inner ear, auditory signal transduction, and the maintenance of adhesion via interactions with fibronectin in the retinal basement membrane. Mutations in the USH2A gene disrupt the normal development and function of hair cells, impair fibronectin assembly, and compromise the adhesive properties of the retinal basement membrane, leading to hearing loss and RP symptoms. Currently, there are no effective therapies for Usher syndrome. Ongoing research focuses on elucidating the genetic mechanisms underlying the disorder and developing gene-based therapeutic strategies. While gene therapy remains preclinical, promising advances have been made with antisense oligonucleotides (ASO) and CRISPR-based gene-editing technologies. QR-421a, an RNA-based oligonucleotide therapy developed by ProQR Therapeutics, targets exon 13 mutations in the USH2A gene associated with USH and non-syndromic RP. This therapeutic approach aims to restore Usherin expression by correcting exon 13 deletions through exon skipping.

Given the focus of ASO and CRISPR therapies on the human USH2A gene, developing humanized mouse models is critical to advancing gene therapies toward clinical applications. Exon 13 of the USH2A gene harbors a hotspot for pathogenic mutations associated with USH, including two common mutations, c.2299delG and c.2276G>T, which are the subject of several therapeutic investigations [2-4, 6]. The B6-hUSH2A(E10-15) mouse model, in which the corresponding mouse Ush2a gene sequence was replaced with human USH2A exons 10 to 15 and their flanking regions, provides a valuable tool for studying USH pathogenesis and evaluating preclinical treatments. Homozygous B6-hUSH2A(E10-15) mice are viable and fertile, making them suitable for drug evaluation and disease modeling. In addition, based on the independently developed TurboKnockout fusion BAC recombination technology, Cyagen can also generate hot mutation models based on this strain and provide customized services for specific mutations to meet the experimental needs in pharmacology and other fields.

Strain Strategy

The start codon (ATG) of the mouse Ush2a gene is located in exon 1, whereas the start codon (ATG) of the human USH2A gene resides in exon 2. Consequently, exons 10–15 of the human USH2A gene correspond to exons 9–14 of the mouse Ush2a gene.


Figure 1. Schematic representation of the gene-editing strategy for the B6-hUSH2A(E10-15) mouse model. Exons 9 to 14 of the mouse Ush2a gene and their flanking sequences were replaced by exons 10 to 15 of the human USH2A gene and their respective flanking sequences.

Application

  • Investigation of the pathogenic mechanisms underlying Usher syndrome (USH) and preclinical evaluation of therapeutic drugs;
  • Development, screening, and preclinical evaluation of drugs targeting USH2A.

Validation Data

1. Expression of human USH2A and mouse Ush2a genes


Figure 2. Gene expression analysis in the eyes of 6-week-old homozygous B6-hUSH2A(E10-15) and wild-type (WT) mice (n=4). RT-qPCR demonstrated significant expression of human USH2A in the eyes of B6-hUSH2A(E10-15) mice, while mouse Ush2a expression was undetectable. In wild-type mice, only the mouse Ush2a gene was expressed (ND: Not detected).

2. Sequencing of USH2A (Exon 10-15) cDNA


Figure 3. Sequencing results of human USH2A exons 10 to 15 in B6-hUSH2A(E10-15) mice. mRNA-derived USH2A cDNA was sequenced, confirming the successful replacement of mouse Ush2a exons 9 to 14 with human USH2A exons 10 to 15. The nucleotide sequence in this region matched the reference sequence of the human USH2A gene.

3. Normal retinal morphology and vasculature in B6-hUSH2A(E10-15) mice


Figure 4. Fundus morphology, OCT, and FFA results of WT and B6J-hUSH2A(E10-15) mice. The fundus morphology, retinal OCT, and fundus fluorescein angiography results of heterozygous and homozygous B6J-hUSH2A(E10-15) mice were consistent with those of WT.

4. Normal photoreceptor function in B6-hUSH2A(E10-15) mice

 

Figure 5. Electroretinogram (ERG) detection results of WT and B6J-hUSH2A(E10-15) Mice. Compared with WT, the amplitudes of the a-wave and b-wave in both scotopic and photopic ERG recordings of heterozygous and homozygous B6J-hUSH2A(E10-15) Mice were nearly identical to those of the WT. The retinal photoreceptor function of heterozygous and homozygous B6J-hUSH2A(E10-15) Mice were normal.

Expanded Information: The Rare Disease Data Center (RDDC)

1. Basic information about the USH2A gene

https://rddc.tsinghua-gd.org/gene/7399


2. USH2A clinical variants

 

3. Disease introduction

Usher syndrome, also known as hereditary deafness-pigmentary retinopathy syndrome, retinitis pigmentosa-deafness syndrome, etc., is a rare autosomal recessive genetic disease with genetic heterogeneity. Patients usually present with congenital sensorineural deafness, progressive retinitis pigmentosa, visual impairment, etc. Usher syndrome is the most common disease that causes deafness and blindness. The incidence rate is 1/5,000 to 1/16,000. According to the age of onset, whether it affects hearing, and whether it affects vestibular function, the disease can be divided into three types: Usher syndrome type I (USH1), type II (USH2), and type III (USH3). Patients with type I usually show almost complete deafness and vestibular dysfunction in childhood, and generally develop symptoms of retinitis pigmentosa (RP) before adulthood; patients with type II have moderate to severe hearing loss without vestibular dysfunction, and develop RP symptoms after adulthood; patients with type III have normal hearing at birth, and gradually develop RP symptoms and hearing loss. Among them, USH1 is the most severe, USH2 is the most common, accounting for about 40%-50% each, and USH3 is the rarest. Considering the clinical features and underdiagnosis of USH2, its incidence rate may be higher than the current estimate.

4. USH2A gene and mutations

USH2A is the most common pathogenic gene in USH2 type, and about 75%~90% of USH2 patients carry pathogenic mutations in the USH2A gene [1]. The USH2A gene encodes usherin protein, which contains laminin EGF-like domains, pentapeptide repeats, and many fibronectin type III domains. Usherin is expressed in the basement membrane and may play an important role in the development and homeostasis of the inner ear and retina. Usherin participates in the development and maintenance of inner ear hair cells and the transmission of auditory signals. In the basement membrane of the retina, USH2A may act as a structural protein to interact with fibronectin and regulate the adhesion of the basement membrane. Mutations in the USH2A gene can affect the development of inner ear hair cells, leading to abnormal assembly of fibronectin in the basement membrane of the retina, weakening the adhesion, and causing hearing loss and RP symptoms.

USH2A pathogenic mutations are relatively concentrated. For example, the c.2299delG mutation in exon 13 has a high incidence rate of up to 50% in the European and American families investigated. Due to frameshift and premature termination codons, this mutation produces an 85% truncated protein of normal transcript size, leading to the loss of Usherin protein function. Research by Japanese scholars shows that the c.8559-2A>G mutation in the USH2A gene may be one of the hot mutations in Asian populations [2-4].

5. Function of non-coding DNA sequences

In 2017, the Fuster-García laboratory achieved in vitro gene editing for the common intron mutation c.2276G>T, p.Cys759Phe (rs80338902) in USH2A gene in USH patients-derived fibroblast models [5].

6. USH2A-targeted gene therapy

Currently, there is no effective treatment for Usher syndrome. Researching the pathogenic genes and developing gene therapy methods are the future focus. Although gene therapy is still in the preclinical stage, various types of pipelines such as antisense oligonucleotide (ASO) drugs and CRISPR gene therapy have been laid out. Humanized mice are often used as experimental subjects. QR-421a, developed by ProQR Therapeutics, is an RNA-based oligonucleotide drug designed to treat Usher syndrome and non-syndromic retinitis pigmentosa (RP) caused by mutations in exon 13 of the USH2A gene. The drug aims to restore normal expression of usherin protein by exon skipping. The pipeline uses induced pluripotent stem cells (iPSCs) from Usher syndrome patients carrying the c.2299delG homozygous mutation as an in vitro research model [6]. The EDIT-102 pipeline developed by Editas Medicine uses adeno-associated virus (AAV) to deliver CRISPR-Pro to target and excise the c.2299delG mutation in exon 13. Partially humanized mice carrying pathogenic mutations (replacing mouse Ush2a gene exon 12 with human USH2A gene exon 13 and introducing the c.2299delG mutation) are used as experimental subjects [7]. Considering the pathogenic mechanism, genetic distribution, and pharmacological research of Usher syndrome, developing humanized mouse models carrying mutations is of great significance for in vivo research of gene therapy drugs.

7. Summary

The USH2A gene is an important pathogenic gene for Usher syndrome. USH2A humanized mice from Cyagen can be used for preclinical research, and customized services can also be provided for different point mutations.

 


References

[1] 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.
[2] 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.
[3] 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.
[4] 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. doi: 10.1086/321269. Epub 2001 Jun 8. Erratum in: Am J Hum Genet 2001 Oct;69(4):922.
[5] Fuster-García C, García-García G, González-Romero E, Jaijo T, Sequedo MD, Ayuso C, Vázquez-Manrique RP, Millán JM, Aller E. USH2A Gene Editing Using the CRISPR System. Mol Ther Nucleic Acids. 2017 Sep 15;8:529-541.
[6] 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.
[7] In Vivo Proof of Concept for EDIT-102: A CRISPR/Cas9-Based Experimental Medicine for USH2ARelated Inherited Retinal Degeneration Caused by Mutations in Exon 13.
https://www.editasmedicine.com/wp-content/uploads/2020/05/Mukherjee_ASGCT-2020-USH2A-poster_final.pdf