B6-hCOL7A1 Mice

Catalog Number: C001428

Strain Name: C57BL/6NCya-Col7a1^tm1(hCOL7A1)/Cya

Genetic Background: C57BL/6NCya

Reproduction: Homozygote x Homozygote

One of Cyagen's HUGO-GT™ (Humanized Genomic Ortholog for Gene Therapy) Mouse Strains

Strain Description

Epidermolysis bullosa (EB) is a hereditary skin disease characterized by the formation of blisters and bullae on the skin and mucous membranes after minor trauma or friction. Common clinical symptoms include blisters, blood blisters, and erosion on the skin. According to the different sites of onset, hereditary EB can be divided into three types: Epidermolysis Bullosa Simplex (EBS), Junctional Epidermolysis Bullosa (JEB), and Dystrophic Epidermolysis Bullosa (DEB). Mutations in the COL7A1 gene are the cause of Dystrophic Epidermolysis Bullosa (DEB), and the different clinical phenotypes presented by DEB are related to the mutation sites and forms of the COL7A1 gene. The COL7A1 gene encodes type VII collagen, which forms anchoring fibrils that bind dermal tissue to epidermal tissue. Functional anchoring fibril deficiency caused by COL7A1 mutations makes the patient’s skin extremely fragile and easily blistered or torn due to minor friction or trauma. At present, at least 324 pathogenic mutations of the COL7A1 gene related to DEB have been found, including nonsense, missense, deletion, insertion, splicing, and regulation ^[1].

The current DEB treatment pipeline is mainly based on gene therapy and small nucleic acid drugs, including ASO drugs, siRNA drugs, and gene therapy based on CRISPR and AAV vector delivery. Among them, COL7A1 is the most important therapeutic target. B-Vec, developed by Krystal Biotech delivers functional COL7A1 genes to skin cells of DEB patients with COL7A1 mutations through HSV-1 vectors to produce functional proteins to promote wound healing, was the first approved gene therapy drug for the DEB. In addition, since most ASO, siRNA, and CRISPR-based therapies target human COL7A1 genes, considering the genetic differences between animals and humans, humanizing mouse genes will help promote further clinical translation of therapies targeting COL7A1. This strain is a mouse Col7a1 gene humanized model and can be used for research on DEB. The homozygous B6-hCOL7A1 mice are viable and fertile. Leveraging its proprietary 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 related to EB.

Strain Strategy

Figure 1. Diagram of the gene editing strategy of B6-hCOL7A1 mice. The sequence from ~3k upstream of exon 1 to 3’UTR of mouse Col7a1 will be replaced with the sequence from ~3k upstream of exon 1 to 3’UTR of human COL7A1 by TurboKnockout targeting technology.

Application

• Research on Epidermolysis bullosa (EB).
  
  

Validation Data

1. Detection of human COL7A1 gene expression

Figure 2. Detection of human COL7A1 gene expression in the spleen and skeletal muscle of wild-type mice (WT) and B6-hCOL7A1 mice (hCOL7A1). The RT-qPCR analysis results showed that the human COL7A1 gene was expressed in both the spleen and skeletal muscle of B6-hCOL7A1 mice, while there was no expression of the human COL7A1 gene in WT mice.

ND: Not detected

2. Detection of mouse Col7a1 gene expression

Figure 3. Detection of mouse Col7a1 gene expression in the spleen and skeletal muscle of wild-type mice (WT) and B6-hCOL7A1 mice (hCOL7A1). The RT-qPCR analysis results showed that the mouse Col7a1 gene was expressed in both the spleen and skeletal muscle of WT mice, while there was no expression of the mouse Col7a1 gene in B6-hCOL7A1 mice.

Expanded Information: The Rare Disease Data Center (RDDC)

1. Basic information about the COL7A1 gene

https://rddc.tsinghua-gd.org/en/gene/1294

2. COL7A1 clinical variants

3. Disease introduction

Epidermolysis bullosa (EB) is a genetic skin disease characterized by the formation of blisters and bullae on the skin and mucous membranes after minor trauma or friction. Common clinical symptoms include the appearance of blisters, blood blisters, and erosion on the skin. Inherited EB can be divided into three categories based on the location of the disease: simplex EB (EBS), junctional EB (JEB), and dystrophic EB (DEB). Among them, the COL7A1 gene is a pathogenic gene related to dystrophic epidermolysis bullosa (DEB), and the different clinical phenotypes presented by DEB are related to the different sites and forms of mutations in the COL7A1 allele. At present, at least 324 pathogenic mutations in the COL7A1 gene related to DEB have been discovered, including 43 nonsense, 127 missense, 65 deletion, 28 insertion, 9 insertion-deletion mutations, 51 splice-site mutations, and 1 regulatory mutation.

4. COL7A1 gene and mutations

The COL7A1 gene encodes collagen type VII, a protein that forms anchoring fibrils that bind the dermal tissue to the epidermal tissue. A lack of functional anchoring fibrils can result in extremely fragile skin that is prone to blistering or tearing from minor friction or trauma.

Mutations in the COL7A1 gene that cause DEB are distributed across several hotspots, with exon 73 being one of them. In Spain, a highly prevalent frameshift mutation is found in exon 80 of DEB patients. c.6527insC is a highly prevalent homozygous mutation, where the insertion mutation causes a premature stop codon, accounting for 46% of the alleles in the Spanish population of recessive DEB patients. Most recessive DEB patients have two different compound heterozygous mutations on each chromosome ^[2-3].

5. Function of non-coding DNA sequences

Intronic mutations in the COL7A1 gene can cause disease. Endogenous 5’ trans-splicing repair can correct pathogenic mutations within the COL7A1 gene ^[4].

6. COL7A1-targeted gene therapy

Drugs targeting COL7A1 mainly include gene therapy drugs such as ASO, siRNA, CRISPR, and AAV delivery. The pipeline of gene therapy drugs for COL7A1 is extensive and diverse. For example, B-Vec from Krystal Biotech is a non-invasive gene therapy drug that delivers the COL7A1 gene using HSV-1 through a gel application, providing patients with two normal functional copies of the COL7A1 gene ^[5]. Another example is ProQR’s pipeline drug QR-313, an ASO drug based on exon 73 skipping the COL7A1 gene ^[6]. In addition, there have been reports of using CRISPR to repair mutations in the COL7A1 gene. This research targets the c.6527dupC mutation and uses a humanized skin mouse model to explore its efficacy ^[7].

In summary, the COL7A1 gene is an important pathogenic gene in epidermolysis bullosa (EB). The treatment of EB mainly focuses on gene therapy, and humanized mice are used to conduct preclinical drug trials. COL7A1 whole-genome humanized model and hot mutation model built on this model from Cyagen can be used for preclinical research on gene therapy for EB. Furthermore, Cyagen can provide customized services for different point mutations of the COL7A1 gene.

References
[1]Dang N and Murrell DF. Mutation Analysis and Characterization of COL7A1 Mutations in Dystrophic Epidermolysis Bullosa. Exp Dermatol 2008;17(7) 553-568.
[2]García M, Bonafont J, Martínez-Palacios J,et al. Preclinical model for phenotypic correction of dystrophic epidermolysis bullosa by in vivo CRISPR-Cas9 delivery using adenoviral vectors.[J].Mol Ther Methods Clin Dev. 2022
[3]Turczynski,Sandrina,Tonasso,et al.Targeted Exon Skipping Restores Type VII Collagen Expression and Anchoring Fibril Formation in an In Vivo RDEB Model[J].The Journal of investigative dermatology, 2016.
[4]Mayr E, Ablinger M, Lettner T, et al. 5'RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa[J].Int J Mol Sci. 2022
[5]In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial[J].Nature Medicine[2023-07-13].DOI:10.1038/s41591-022-01737-y.
[6]Bornert O , Hogervorst M , Nauroy P ,et al.QR-313, an antisense oligonucleotide, shows therapeutic efficacy for treatment of dominant and recessive dystrophic epidermolysis bullosa: a preclinical study[J]. Journal of Investigative Dermatology, 2020.DOI:10.1016/j.jid.2020.08.018.
[7]Hainzl S , Peking P , Kocher T , et al.COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.[J]. Molecular Therapy the Journal of the American Society of Gene Therapy, 2017, 25(11).DOI:10.1016/j.ymthe.2017.07.005.