B6-hABCA4 Mice

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Catalog Number: C001551

Strain Name: C57BL/6JCya-Abca4tm1(hABCA4)/Cya

Genetic Background: C57BL/6JCya

Reproduction: Homozygote x Homozygote

One of Cyagen’s HUGO-GTTM (Humanized Genomic Ortholog for Gene Therapy) Strains

 

Strain Description

Stargardt Disease (STGD) is a hereditary macular dystrophy marked by yellowish fusiform spots in the retinal pigment epithelium, leading to macular atrophy. It primarily affects children and adolescents, causing progressive central vision loss and mild color vision impairment. The fundus may show pale yellow lesions with gold foil-like reflections and yellow-white spots around the posterior pole. Advanced stages involve atrophy of the retinal pigment epithelium, photoreceptor cells, and choriocapillaris. STGD is also common in sporadic cases and more frequent in children of consanguineous marriages. It affects both eyes bilaterally and progresses synchronously without significant gender differences, with an incidence of approximately 1/8000 to 1/13000. STGD is an autosomal recessive retinal disease caused by ABCA4 gene mutations, accounting for 95% of cases.

The ABCA4 gene encodes a retina-specific ABC transporter protein that removes retinal derivatives and toxic metabolites after rhodopsin photobleaching. Mutations in ABCA4 lead to the accumulation of these substances, causing apoptosis of retinal pigment epithelial and photoreceptor cells, resulting in retinal degenerative diseases. ABCA4 mutations are linked to Stargardt Disease (STGD), Cone-rod Dystrophy (CRD), and Retinitis Pigmentosa (RP). The clinical phenotype depends on the extent of ABCA4 mutations, with severe and mild mutations or two moderate mutations predisposing to STGD, and one moderate mutation predisposing to CRD.

Currently, the drug pipeline for treating Stargardt disease (STGD) primarily focuses on supplemental delivery methods for ABCA4-targeted drugs. Among them, ProQR has developed a therapeutic antisense oligonucleotide (ASO) drug, QR-1011, which targets the c.5461-10T>C mutation [1]. Most ASO medicines and gene therapies act on the human ABCA4 gene. Considering the genetic differences between animals and humans, modifying mouse genes to be more human-like would help accelerate gene therapies targeting ABCA4 into the clinical stage. This strain is a mouse Abca4 gene humanized model and can be used to research STGD, CRD, and RP. The homozygous B6-hABCA4 mice are viable and fertile. In addition, based on the independently developed TurboKnockout fusion BAC recombination technology, Cyagen can also generate hot mutation (ABCA4 c.5461-10 T to C) models based on this strain and provide customized services for specific mutations to meet the experimental needs in pharmacology and other fields.

Strain Strategy


Figure 1. Gene editing strategy of B6-hABCA4 mice. The sequences from the ATG start codon to the TGA stop codon of the mouse Abca4 gene were replaced with the sequences from the ATG start codon to the TGA stop codon of the human ABCA4 gene.

Application

  • Research on Stargardt Disease (STGD);
  • Research on Cone-rod Dystrophy (CRD);
  • Research on Retinitis Pigmentosa (RP);

Validation Data

1. Expression of human ABCA4 and mouse Abca4 genes


Figure 2. Gene expression analysis in the eyes of 4-week-old homozygous B6-hABCA4 and wild-type (WT) mice. RT-qPCR demonstrated significant expression of human ABCA4 in the eyes of B6-hABCA4 mice, while mouse Abca4 expression was undetectable. In wild-type mice, only the mouse Abca4 gene was expressed (ND: Not detected).

2. Sequencing of ABCA4 cDNA


Figure 3. Sequencing results of the human ABCA4 gene exons 38 to 41 (Exon 38-41) in B6-hABCA4 mice. cDNA was obtained by reverse transcription amplification using primers specifically paired with human ABCA4 mRNA, followed by gel electrophoresis to confirm the expression of the human ABCA4 gene. The results showed the presence of human ABCA4 cDNA bands in the eye tissue of B6-hABCA4 mice, with band sizes matching expectations and no abnormal transcription bands. Sequencing analysis of the ABCA4 cDNA revealed that exons 38 to 41 of the mouse Abca4 gene were successfully replaced with the corresponding human ABCA4 gene exons, with nucleotide sequences identical to the human ABCA4 reference sequence.

3. Normal retinal morphology and vasculature in B6-hABCA4 mice


Figure 4. Fundus morphology, OCT, and FFA results of wild-type and B6J-hABCA4 mice. The fundus morphology, retinal OCT, and fundus fluorescein angiography results of B6J-hABCA4 mice were consistent with those of WT.

4. Electroretinogram (ERG)

 

Figure 5. Electroretinogram (ERG) detection results of WT and B6J-hABCA4 mice. Compared with the wild-type, the amplitudes of the a-wave and b-wave in both scotopic and photopic ERG recordings of homozygous B6J-hABCA4 mice were nearly identical to those of the WT. The retinal photoreceptor function of B6J-hABCA4 mice was normal.

Expanded Information: The Rare Disease Data Center (RDDC)

1. Basic information about the ABCA4 gene

https://rddc.tsinghua-gd.org/gene/24

2. ABCA4 clinical variants

3. Disease introduction

Stargardt Disease (STGD) is a hereditary macular dystrophy marked by yellowish fusiform spots in the retinal pigment epithelium, leading to macular atrophy. It primarily affects children and adolescents, causing progressive central vision loss and mild color vision impairment. The fundus may show pale yellow lesions with gold foil-like reflections and yellow-white spots around the posterior pole. Advanced stages involve atrophy of the retinal pigment epithelium, photoreceptor cells, and choriocapillaris. STGD is also common in sporadic cases and more frequent in children of consanguineous marriages. It affects both eyes bilaterally and progresses synchronously without significant gender differences, with an incidence of approximately 1/8000 to 1/13000. STGD is an autosomal recessive retinal disease caused by ABCA4 gene mutations, accounting for 95% of cases.

4. ABCA4 gene and mutations

The ABCA4 gene encodes a retina-specific ABC transporter protein that removes retinal derivatives and toxic metabolites after rhodopsin photobleaching. Mutations in ABCA4 lead to the accumulation of these substances, causing apoptosis of retinal pigment epithelial and photoreceptor cells, resulting in retinal degenerative diseases. ABCA4 mutations are linked to Stargardt Disease (STGD), Cone-rod Dystrophy (CRD), and Retinitis Pigmentosa (RP). The clinical phenotype depends on the extent of ABCA4 mutations, with severe and mild mutations or two moderate mutations predisposing to STGD, and one moderate mutation predisposing to CRD.

The ABCA4 gene contains 50 exons and spans approximately 128 kb of genomic DNA. It encodes a protein consisting of 2,273 amino acids, with a molecular weight of approximately 250 kDa. Mutations in the ABCA4 gene exhibit distinct racial specificity, and compound mutations are a characteristic feature of ABCA4 gene mutations. To date, over 2,000 variants have been identified, including missense, nonsense, splicing, structural, and deep intronic variants. Missense variants account for the majority of ABCA4 variants. The most common severe pathogenic mutation is c.5461-10T>C. According to reports, this mutation has a higher frequency in South African patients [2].

5. Function of non-coding DNA sequences

The most common pathogenic mutation is a c.5461-10T>C mutation in intron 38 [3], which can cause splicing defects, leading to a reduction in full-length mRNA of fibroblasts and skipping of exons 39-40. Compared with the control group without mutations, a decrease in full-length ABCA4 protein levels can be observed. Most ABCA4 gene mutations are non-classical splicing site mutations [4].

6. ABCA4-targeted gene therapy

Current treatments for STGD primarily focus on ABCA4-targeted supplemental delivery therapies. ProQR has developed a therapeutic antisense oligonucleotide (ASO) drug, QR-1011, which targets the c.5461-10T>C mutation, using organoids carrying this mutation as a preclinical model [1]. Sanofi’s SAR422459 (EIAV-ABCA) pipeline delivers ABCA4 via a lentiviral vector system to Abca4−/− mouse models to treat STGD. While delivery therapies dominate current STGD treatments, targeting the reduction of endogenous ABCA4 mutation expression also holds promise, given the disease’s pathogenic mechanism involves ABCA4 mutations causing toxicity.

7. Summary

The ABCA4 gene is an important pathogenic gene for various retinal degenerative diseases such as Stargardt disease (STGD), Cone-rod Dystrophy (CRD), and Retinitis Pigmentosa (RP). ABCA4 humanized mice and the hotspot mutation disease model (ABCA4 c.5461-10 T to C) based on this strain from Cyagen can be used for preclinical research on STGD, CRD, and RP, and customized services can also be provided for different point mutations.



References

[1] Kaltak M, de Bruijn P, Piccolo D, et al. Antisense oligonucleotide therapy corrects splicing in the common Stargardt disease type 1-causing variant ABCA4 c.5461-10T>C. Mol Ther Nucleic Acids. 2023 Feb 18;31:674-688. doi: 10.1016/j.omtn.2023.02.020.
[2] Roberts L J , Nossek C A , Greenberg L J ,et al.Stargardt macular dystrophy: common ABCA4 mutations in South Africa—establishment of a rapid genetic test and relating risk to patients[J].Molecular Vision, 2012, 18(31-33):280-289.DOI:doi:10.3928/1081597X-20111209-01.
[3] Aukrust, IngvildJansson, Ragnhild W.Bredrup, CecilieRusaas, Hilde E.Berland, SirenJorgensen, AgneteHaug, Marte G.Rodahl, EyvindHouge, GunnarKnappskog, Per M.The intronic ABCA4 c.5461-10T > C variant, frequently seen in patients with Stargardt disease, causes splice defects and reduced ABCA4 protein level[J]. Acta ophthalmologica, 2017,
[4] ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease[J]. Genome Research, 2018.