Hope for "Butterfly Children"—New Models in Research on Dystrophic Epidermolysis Bullosa (DEB)

In everyday life, hugging is a way for people to express love and kindness. However, for individuals with Epidermolysis Bullosa (EB), this simple act of intimacy can cause immense pain. EB is a rare genetic skin disorder that causes fragile, blistering skin that is recognized as one of the most painful conditions known to humanity. The skin of EB patients is as fragile as butterfly wings – with even a light touch causing it to peel off like butterfly dust – which can lead to severe systemic complications. Because of this, EB patients are often referred to as "Butterfly Children." The recent FDA approval of Vyjuvek in May 2023 indicates the beginning of effective gene therapy treatments for disorders such as EB and its severe subtype, Dystrophic Epidermolysis Bullosa (DEB).

Today we introduce several Dystrophic Epidermolysis Bullosa (DEB) research models independently developed by Cyagen to support preclinical research seeking to develop effective gene therapy drugs.

Pathogenic Mechanisms of Dystrophic Epidermolysis Bullosa (DEB)

Dystrophic Epidermolysis Bullosa (DEB) is a major and severe subtype of Epidermolysis Bullosa (EB), accounting for approximately 25% of all EB cases. The skin covers the body surface and consists of the following layers, from outermost to innermost: epidermis, dermis, and subcutaneous. DEB is caused by mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein closely related to the formation of anchoring fibrils (AF) that bind the dermis and epidermis together.^[1-2] DEB has two genetic patterns: dominant (DDEB) and recessive (RDEB). Typically, RDEB is more severe than DDEB. Symptoms of RDEB include fragile skin, widespread wounds, and extracutaneous manifestations affecting the mouth, eyes, gastrointestinal tract, and genitourinary system. Given that RDEB often begins in the neonatal period, patients frequently succumb to infections. Patients with RDEB have a lifetime risk above 90% of developing aggressive squamous cell carcinoma.^[1-2] 

Figure 1: Mechanisms, Classification, Affected Areas, and Symptoms of Dystrophic Epidermolysis Bullosa (DEB) ^[2]

Development of DEB Therapies and Mouse Models

Research on DEB treatments primarily focuses on correcting COL7A1 gene mutations or supplementing gene copies.^[3] This includes using gene editing technologies like CRISPR to repair COL7A1 mutations,^[4-6] regulating COL7A1 splicing patterns with antisense oligonucleotides (ASOs), ^[7] and delivering COL7A1 gene copies via vectors such as AAV or HSV-1.^[8-9] These therapies require initial validation in animal models.

In humans, RDEB is caused by homozygous null mutations in COL7A1, and similar mechanisms are observed in mouse models. For example, Col7a1 homozygous knockout mice exhibit high mortality rates after birth, developing hemorrhagic blisters on the paw pads within 24-48 hours, followed by severe RDEB symptoms.^[10] This knockout mouse model is commonly used for preclinical testing of RDEB supplement therapies. However, in the fields of gene editing and antisense oligonucleotide (ASO) research, the lack of suitable mouse models to precisely target humanized gene sequences poses a significant challenge. Gene therapy-related in vivo studies require the ability to precisely target humanized gene sequences, presenting the need for a mouse model with a full-length genomic DNA humanized mice. These models can faithfully replicate human gene expression patterns, regulations, and functional properties in a mouse model.

Figure 2: Research on Therapeutic Strategies for Dystrophic Epidermolysis Bullosa (DEB) ^[3]

The HUGO-GT™ Next-Generation Humanized Mouse Models program is specifically designed for gene therapy and rare disease research, featuring full-length genomic sequence humanized mouse models and patient-specific pathogenic point mutation disease models. Cyagen has independently developed the B6-hCOL7A1 humanized mouse model (Product No.: C001428) and introduced the common recurrent pathogenic mutation c.6527dupC to construct the B6-hCOL7A1*c.6527dupC disease model (Product No.: C001538).^[11] This model aims to meet the research needs of gene editing and small nucleic acid therapies. Additionally, a Col7a1 gene knockout (KO) mouse model (Product No.: C001539) has been developed for the field of supplement therapies. Both B6-hCOL7A1*c.6527dupC mice and Col7a1 KO mice exhibit significant RDEB phenotypes. The detailed phenotype information for these models is provided below.

Gene Expression Analysis

In homozygous Col7a1 KO mice, the expression of the murine Col7a1 gene is completely knocked out. Similarly, homozygous B6-hCOL7A1*c.6527dupC mice lack murine Col7a1 gene expression but can express human COL7A1 gene at equivalent levels. Although the c.6527dupC mutation does not affect gene transcription, it results in abnormal protein expression.

Figure 3: Expression Analysis of Endogenous Mouse Col7a1 Gene and Human COL7A1 Gene

Mice Lack Functional COL7A1 Protein Expression

Wild-type mice and heterozygous Col7a1 KO mice express murine COL7A1 protein, but homozygous Col7a1 KO mice do not express this protein. Similarly, homozygous B6-hCOL7A1 mice and heterozygous B6-hCOL7A1*c.6527dupC mice express human COL7A1 protein, while homozygous B6-hCOL7A1*c.6527dupC mice do not express the protein. Both homozygous B6-hCOL7A1*c.6527dupC mice and homozygous Col7a1 KO mice exhibit separation of the epidermis and dermis in skin tissue.

Figure 4: Immunohistochemical Detection of COL7A1 Protein Expression in the Skin of Col7a1 knockout (KO) Mice and B6-hCOL7A1*c.6527dupC Mice

Mice Exhibit Skin Erythema and Blistering Phenotype

Homozygous Col7a1 KO mice and homozygous B6-hCOL7A1*c.6527dupC mice both exhibit skin erythema and blistering symptoms on the first day after birth. Among them, Col7a1 KO mice had more severe symptoms: with the phenotype primarily affecting the front and back paw pads, and dying within three days of birth.

Figure 5: Col7a1 KO Mice and B6-hCOL7A1*c.6527dupC Mice Exhibit Skin Erythema and Blistering Phenotype

Pathological Progression of Skin in B6-hCOL7A1*c.6527dupC Mice

On the first day after birth, homozygous B6-hCOL7A1*c.6527dupC mice exhibit erythema and blistering on the front and hind paws. By the second day, erythema and blistering are observed on the tail, in addition to the paws. By the seventh day, erythema and blistering are no longer visible, but large areas of skin peeling appear.

Figure 6: Progression of Skin Erythema and Peeling Phenotype in B6-hCOL7A1*c.6527dupC Mice

Mice Exhibit Pathological Separation Of The Epidermis And Dermis

Homozygous Col7a1 KO mice and homozygous B6-hCOL7A1*c.6527dupC mice both exhibit significant subcutaneous edema (indicated by green asterisks in the figure) and observable separation of the epidermis and dermis.

Figure 7: H&E Staining of Skin Tissue in Col7a1 KO Mice and B6-hCOL7A1*c.6527dupC Mice

Conclusion

Homozygous Col7a1 KO mice (Product No.: C001539) and homozygous B6-hCOL7A1*c.6527dupC mice (Product No.: C001538) both exhibit the typical symptoms of Epidermolysis Bullosa (EB), including skin erythema, blistering, subcutaneous edema, and separation of the epidermis and dermis. The symptoms are more severe in Col7a1 KO mice, primarily affecting the fore and hind paw pads, with these mice dying within three days after birth. In contrast, B6-hCOL7A1*c.6527dupC mice exhibit relatively milder symptoms, starting with erythema and blistering on the paws and progressing to large areas of skin peeling by the seventh day. Additionally, skin from homozygous B6-hCOL7A1 mice and heterozygous B6-hCOL7A1*c.6527dupC mice expresses the COL7A1 protein, whereas homozygous B6-hCOL7A1*c.6527dupC mice and homozygous Col7a1 KO mice do not express this protein. These humanized and knockout (KO) mouse models provide essential tools for preclinical DEB research, aiding in a deeper understanding of the pathogenesis of this rare disease and offering potential avenues for developing new treatments.

Additionally, Cyagen offers a variety of humanized and point mutation disease models for gene therapy and rare disease research, catering to the experimental needs of researchers in these fields.

HUGO-GT™ Next-Generation Humanized Models

References:
[1] Eichstadt et al., "From Clinical Phenotype to Genotypic Modelling: Incidence and Prevalence of Recessive Dystrophic Epidermolysis Bullosa (RDEB)", Clin Cosmet Investig Dermatol, vol. 12, pp. 933-942, 2019.
[2] DEBRA International, "About EB: EB in Depth", Retrieved May 17, 2024, from https://www.debra.org/about-eb/eb-depth.
[3] Hou et al., "Innovations in the Treatment of Dystrophic Epidermolysis Bullosa (DEB): Current Landscape and Prospects", Ther Clin Risk Manag, vol. 19, pp. 455-473, 2023.
[4] Bonafont et al., "Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing", Mol Ther, vol. 29, no. 6, pp. 2008-2018, 2021.
[5] Hainzl et al., "COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa", Mol Ther, vol. 25, no. 11, pp. 2573-2584, 2017.
[6] García et al., "Preclinical model for phenotypic correction of dystrophic epidermolysis bullosa by in vivo CRISPR-Cas9 delivery using adenoviral vectors", Mol Ther Methods Clin Dev, vol. 27, pp. 96-108, 2022.
[7] Bornert et al., "QR-313, an Antisense Oligonucleotide, Shows Therapeutic Efficacy for Treatment of Dominant and Recessive Dystrophic Epidermolysis Bullosa: A Preclinical Study", J Invest Dermatol, vol. 141, no. 4, pp. 883-893.e6, 2021.
[8] Gurevich et al., "In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial", Nat Med, vol. 28, no. 4, pp. 780-788, 2022.
[9] Chamorro et al., "Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes", Mol Ther Nucleic Acids, vol. 5, no. 4, e307, 2016.
[10] Fritsch et al., "A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy", J Clin Invest, vol. 118, no. 5, pp. 1669-79, 2008.
[11] Sanchez-Jimeno et al., "Recessive dystrophic epidermolysis bullosa: the origin of the c.6527insC mutation in the Spanish population", Br J Dermatol, vol. 168, no. 1, pp. 226-9, 2013.