How the B6-hTGFBI Humanized Mouse Model is Advancing Corneal Dystrophy Research

Corneal dystrophy (CD) is a group of rare hereditary disorders caused by abnormal deposits of substances in the cornea. Clinically, CD typically manifests as a gradual loss of corneal transparency in the eyes, leading to recurrent corneal erosion and visual impairment. Granular corneal dystrophy (GCD) is a common subtype of CD, caused by mutations in the Transforming Growth Factor Beta Induced (TGFBI) gene. ^[1] The TGFBI gene encodes the TGFBIp protein, which plays a key role in cell interactions with collagen, contributing to cell adhesion, migration, and proliferation. Mutations in TGFBIp result in abnormal deposits in the corneal epithelium and stroma, reducing corneal transparency and refractive power, ultimately impairing vision. ^[1-2]

Figure 1. Ocular symptoms of a patient with granular corneal dystrophy (GCD), showing deposits located in the anterior stroma of the cornea. ^[2]

The Need for New Corneal Dystrophy (CD) Treatment Strategies

The cornea is a transparent, avascular tissue located at the front of the eye, responsible for focusing light onto the retina and providing two-thirds of the eye's refractive power. The hallmark pathological histological feature of corneal dystrophy (CD) is the accumulation of specific substances in different layers of the cornea, often affecting both eyes. Different types of CD can lead to varying degrees of visual impairment. ^[3] Currently, there is no cure for corneal dystrophy (CD). Corneal transplantation is the primary established treatment, but it faces challenges such as postoperative disease recurrence, transplant failure, complications, and a shortage of donor corneal tissues. As a result, developing novel alternative therapies using gene therapy, regenerative medicine, or cell-based therapies are set to underpin future research. ^[3-4] Since TGFBI-related corneal dystrophy is one of the most common subtypes, corneal dystrophy associated with TGFBI mutations and its targeted therapies have quickly become the primary focus of current research.

Figure 2. Different transport and deposition patterns of normal and mutant TGFBIp proteins. ^[4]

TGFBI-Targeted Therapies: RNA interference and Gene Editing

The TGFBI gene encodes the TGFBIp protein, which plays a key role in various physiological and pathological processes by mediating cell adhesion, migration, proliferation, and differentiation. TGFBIp is expressed in the cornea, skin, and connective tissues, where it connects with collagen to provide structural support to the corneal extracellular matrix. ^[5] It is known that mutations in TGFBI can lead to abnormal corneal deposits, making the inhibition of mutant gene expression a potential therapeutic strategy. SiSaf Ltd has developed two therapies targeting TGFBI mutations: SIS-201-CD, an RNA interference therapy, and SIS-201-CDC, a CRISPR-Pro gene-editing therapy. Both approaches aim to inhibit or edit the mutant TGFBI gene to prevent the production or accumulation of abnormal proteins. ^[6-7] Additionally, recent studies have shown that TGFBI is abnormally expressed in various cancers and exhibits immunosuppressive effects within the tumor immune microenvironment. ^[8] TGFBI plays a crucial role in tumor cell proliferation, angiogenesis, and apoptosis, promoting the invasion and metastasis of multiple types of tumors. ^[8-9] Therefore, TGFBI-targeted therapies also hold potential as an anti-cancer strategy.

Figure 3. Early preclinical cell line studies of gene editing therapy targeting the mutant TGFBI gene. ^[7]

Introducing the B6-hTGFBI Humanized Mouse Model for TGFBI Research

Both RNA interference and gene-editing therapies target human genes, but differences between mouse and human genes can limit their clinical translation. Given the genetic differences between mice and humans, humanizing the mouse gene will help accelerate the clinical translation of TGFBI-targeted gene therapies. To overcome this, Cyagen has successfully developed the B6-hTGFBI humanized mouse model (Product No.: C001546) by replacing the mouse Tgfbi gene sequence with the human TGFBI gene sequence. These mice express the human TGFBI gene, exhibit normal development, and display no abnormal ocular phenotype. Cyagen also plans to develop additional humanized point mutation models based on this strain, providing valuable tools for TGFBI-related research. Any researchers interested are welcome to inquire by contacting us. Below is the phenotypic information for the B6-hTGFBI mouse model.

Key Features of the B6-hTGFBI Mouse Model

Successful Expression of Human TGFBI Gene:

Homozygous B6-hTGFBI mice successfully express the human TGFBI gene in both the liver and eyes, while the mouse Tgfbi gene is not expressed.

Figure 4. Gene expression analysis in the liver and eyes of B6-hTGFBI mice and wild-type (WT) mice.

Normal Ocular/Eye Morphology:

Both homozygous and heterozygous B6-hTGFBI mice show no significant differences in the morphology of various ocular tissues compared to wild-type mice (WT), indicating a normal ocular phenotype in B6-hTGFBI mice.

Figure 5. Fundus, retinal optical coherence tomography (OCT), cornea, and anterior chamber OCT analysis.

Normal Retinal Photoreceptor Function:

Both homozygous and heterozygous B6-hTGFBI mice exhibit a-wave and b-wave amplitudes in scotopic (dark-adapted) and photopic (light-adapted) ERG that are consistent with those of wild-type mice (WT), indicating normal retinal photoreceptor function in these mice.

Figure 6. Electroretinogram (ERG) analysis of wild-type (WT) and B6-hTGFBI mice.

Summary

The B6-hTGFBI mouse model (Product No.: C001546) successfully expresses the human TGFBI gene and no longer expresses the mouse Tgfbi gene. These mice develop normally without abnormal ocular phenotypes. The morphology of their ocular tissues shows no significant differences from that of wild-type mice, and their retinal photoreceptor function is normal. The model’s normal eye morphology and retinal function make it an ideal platform for studying TGFBI-related corneal dystrophy (CD) and can be used for screening and preclinical validation of RNA interference therapies and gene editing therapy candidates.

Additionally, Cyagen is developing humanized point mutation disease models based on this strain. We also offer customized services for different point mutations to meet the needs of preclinical research and development. Below are several of Cyagen’s HUGO-GT™ Next-Generation Humanized Models developed for disease modeling.

HUGO-GT™ Next-Generation Humanized Models

References:

[1] Li Haoliang, Liang Shu. Research progress on TGFBI-related granular corneal dystrophy [J]. Chinese Journal of Clinical Physicians (Electronic Edition), 2020, 14(11): 931-936.
[2]Corneal dystrophies. Nat Rev Dis Primers. 2020 Jun 11;6(1):47. 
[3]Han KE, Choi SI, Kim TI, Maeng YS, Stulting RD, Ji YW, Kim EK. Pathogenesis and treatments of TGFBI corneal dystrophies. Prog Retin Eye Res. 2016 Jan;50:67-88.
[4]Vision Center. (2024). Corneal Dystrophy: Symptoms, Types, and Treatments. https://www.nei.nih.gov/learn-about-eye-health/eye-conditions-and-diseases/corneal-conditions/corneal-dystrophies
[5]Nielsen NS, Poulsen ET, Lukassen MV, Chao Shern C, Mogensen EH, Weberskov CE, DeDionisio L, Schauser L, Moore TCB, Otzen DE, Hjortdal J, Enghild JJ. Biochemical mechanisms of aggregation in TGFBI-linked corneal dystrophies. Prog Retin Eye Res. 2020 Jul;77:100843.
[6]SiSaf Ltd. (2024). Pipeline. https://sisaf.com/pipeline/
[7]Taketani Y, Kitamoto K, Sakisaka T, Kimakura M, Toyono T, Yamagami S, Amano S, Kuroda M, Moore T, Usui T, Ouchi Y. Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair. Sci Rep. 2017 Dec 1;7(1):16713.
[8]Huang H, Tang Q, Li S, Qin Y, Zhu G. TGFBI: A novel therapeutic target for cancer. Int Immunopharmacol. 2024 Jun 15;134:112180.
[9]Corona A, Blobe GC. The role of the extracellular matrix protein TGFBI in cancer. Cell Signal. 2021 Aug;84:110028.