B6-hCOL7A1*c.6527dupC Mice

The c.6527dupC mutation is introduced into the 81st exon of the existing humanized COL7A1 gene in B6-hCOL7A1 mice (Catalog Number: C001428) using gene editing technology.

B6-hCOL7A1*c.6527dupC mice can be used for the mechanistic study of Dystrophic Epidermolysis Bullosa (DEB), as well as the development, screening, and evaluation of therapeutic drugs.

1. Homozygous B6-hCOL7A1*c.6527dupC mice successfully express the human COL7A1 gene

Figure 1. RT-qPCR detection of human COL7A1 and mouse Col7a1 gene expression in wild-type mice (WT), homozygous B6-hCOL7A1 mice, heterozygous B6-hCOL7A1*c.6527dupC mice, and homozygous B6-hCOL7A1*c.6527dupC mice. The detection results show that both homozygous B6-hCOL7A1 mice and B6-hCOL7A1*c.6527dupC mice do not express the mouse Col7a1 gene, but they can express the human COL7A1 gene at an equivalent level (Note: the c.6527dupC mutation does not affect gene transcription, but it causes abnormal protein expression, so there is no expression difference at the mRNA level).
ND: Not detected

 

2. Homozygous B6-hCOL7A1*c.6527dupC mice lack the expression of COL7A1 protein

Figure 2. Immunohistochemical detection of COL7A1 protein expression in homozygous B6-hCOL7A1 wild-type humanized mice (HO), heterozygous B6-hCOL7A1*c.6527dupC mice (HE), and homozygous B6-hCOL7A1*c.6527dupC mice (HO). The data show that both B6-hCOL7A1 mice and heterozygous B6-hCOL7A1*c.6527dupC mice express the COL7A1 protein, while homozygous B6-hCOL7A1*c.6527dupC mice do not. In addition, the skin tissue of homozygous B6-hCOL7A1*c.6527dupC mice also exhibits separation between the epidermis and dermis.

 

3. Homozygous B6-hCOL7A1*c.6527dupC mice exhibit significant subcutaneous edema

Figure 3. H&E staining detection of skin tissue in wild-type mice (WT) and homozygous B6-hCOL7A1*c.6527dupC mice (HO). The data show that compared with wild-type mice, the skin of homozygous B6-hCOL7A1*c.6527dupC mice exhibits significant subcutaneous edema, and a separation between the epidermis and dermis is observed (indicated by the green asterisk).

 

4. Homozygous B6-hCOL7A1*c.6527dupC mice exhibit a phenotype of skin redness and blistering

Figure 4. Appearance monitoring of wild-type mice (WT) and homozygous B6-hCOL7A1*c.6527dupC mice (HO). The data show that compared with wild-type mice, homozygous B6-hCOL7A1*c.6527dupC mice exhibit redness and blistering on the fore/hind paws on the first day after birth, and on the second day, redness and blistering are observed on the fore and hind paws as well as the tail. By the seventh day, no redness or blistering is observed, but there is a large area of skin peeling. In addition, the body size of homozygous B6-hCOL7A1*c.6527dupC mice is significantly smaller than that of wild-type mice, and these mice ultimately die within ten days after birth.

B6-hCOL7A1*c.6527dupC mice successfully express the human COL7A1 gene and do not express the mouse Col7a1 gene. Due to the presence of the c.6527dupC mutation, although the expression of the human COL7A1 gene is normal at the mRNA level, the expression at the protein level is blocked, leading to the absence of COL7A1 protein. The absence of COL7A1 protein triggers a series of phenotypic changes, including the separation of the epidermis and dermis in skin tissue, subcutaneous edema, and skin redness and blistering. In addition, these mice are significantly smaller in size than wild-type mice and die within ten days after birth.

Furthermore, Cyagen Biosciences also offers a Col7a1 KO mouse model (Catalog Number: C001539) , which has a more severe disease phenotype than this strain, and homozygous Col7a1 KO mice die within three days after birth. 

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.