Due to the limited efficacy of traditional treatments, ophthalmic diseases have quickly become a breakthrough point for gene therapy, but many questions about mouse models for preclinical ophthalmology research remain. Although there are many gene therapy drugs currently on the market, basic research and preclinical translation in ophthalmology face many difficulties, such as complicated operation, long production period of experimental models, and expensive equipment.
Cyagen has established an ophthalmic gene therapy platform to overcome the above obstacles. Herein, we answer several questions related to mouse models for preclinical Ophthalmology research.
Cyagen offers a collection of gene-edited and humanized mouse models to study diseases such as retinitis pigmentosa, congenital nystagmus, age-related macular degeneration (AMD), and total color blindness. These models are created using our proprietary TurboKnockout and optimized CRISPR-Pro technologies, and are designed to support the needs of customers in basic research and new drug development. Additionally, we provide personalized cell and animal model customization services for researchers.
Q: What is Optical Coherence Tomography testing and how is the data interpreted?
A: OCT stands for Optical Coherence Tomography, which is a non-contact, high-resolution tomographic imaging and biomicroscopy device. It can be used for in vivo examination, cross-sectional imaging, and measurement of posterior ocular structures (including retina, retinal nerve fiber layer, macula, and optic disc), that helps for the diagnosis of macular holes, cystoid macular edema, diabetic retinopathy, age-related macular degeneration, and glaucoma, among other eye diseases. Most of our current ophthalmic disease models involve phenotypes with outer nuclear layer (ONL) loss and retinal pigment epithelium cell layer (RPE) loss. Below, Figure 1 shows a schematic diagram of retinal layers by OCT examination.
Figure 1. Mouse OCT examination of retinal layers. RNFL/GC: Retinal Nerve Fiber Layer/Ganglion Cell Layer, IP: Inner Plexiform Layer, IN: Inner Nuclear Layer, OP: Outer Plexiform Layer, ON: Outer Nuclear Layer, ELM: External Limiting Membrane, IS/OS: Photoreceptor Inner/Outer Segments, RPE: Retinal Pigment Epithelium Cell Layer.
Q: How to interpret Electroretinogram tests?
A: ERG stands for Electroretinogram, which records a series of combined potential changes in retinal photoreceptor cells elicited by light stimulation through electrodes, and is amplified by a special signal device for further analysis of retinal function. In ERG, the a-wave is a cathodic wave, mainly generated by photoreceptor cells (primarily rod and cone cells), while the b-wave is a positive potential, generated by Müller cells and bipolar cells within the retina.
Q: Which one is used for ERG evaluation? The amplitude or peak height?
A: ERG is evaluated based on amplitude.
Q: How to define amplitude in ERG?
A: As shown in Figure 2, the amplitude of a-wave refers to the difference between baseline and lowest point of a-wave. In contrast, the amplitude of the b-wave is not based on baseline, but defined as the difference between lowest point of a-wave and highest point of b-wave.
Figure 2. Definition of amplitudes of a and b-wave in ERG.
Q: What is the ERG data for Cyagen's ophthalmic disease models?
A: Cyagen's mouse modelIs, including RD1 (C001277 and C001384), RD2 (C001385), RD10 (C001277), Tub-KO (C001386), Rpe65 R44X (C001360), and Rpe65-KO (C001387), are all related to retinitis pigmentosa (RP). According to the literature, most RP diseases exhibit a significant decrease in scotopic ERG. Our current data demonstrates a significant decrease or absence of scotopic ERG in all of the above models.
Q: What are the definitions of early-onset and slow-progressing in RD1 and RD2 mice?
A: Early-onset is defined by age of onset, while slow-progressing is defined by the progression of disease. RD1 mice exhibit severe retinal lesions at two weeks of age, with an extinguished ERG at the same time. RD2 mice show outer nuclear layer disappear at 9 months, while ERG completely extinguished until 12 months. The ERG alteration of RD1 mice happens three weeks after birth, which is an early stage of testing for mice (mice eyes first open around 14 days), indicating that RD1 is an early-onset model. Furthermore, the disease progression of RD2 mice is very slow, and the severity of disease phenotype is far less than RD1 mice at 11 weeks of age, which suggests that RD2 is a slow-progressing model.
Q: What does slow-progressing mean? The disease onset is delayed, or it takes long time to reach certain disease severity, or the level of disease severity is low?
A: Slow-progressing means there is a long disease course to develop severe retinal lesions. However, it can also be known as having a slow progression of disease within a short period of time, and low level of severity at young age.
Q: What are the differences between C001277 and C001384 RD1 mice?
A: C001277 is a spontaneous mutation of the mouse Pde6b gene. The Pde6b gene is essential for the transmission of visual signals, and this mutation leads to severe early-onset retinal degeneration in mice, including rapid apoptosis of rod photoreceptors and outer nuclear layer disappearance, showing complete visual function loss through ERG test within three weeks. C001384 is a Pde6b gene knockout (Pde6b-KO) model, which the protein expression is absent. Similarly, the mice exhibit severe phenotypes of rod photoreceptor apoptosis, outer nuclear layer disappearance, and extinguished ERG. In conclusion, these 2 models share similar disease progression and phenotype severity; however, aspects regarding gene modification methods, timeline and level of disease phenotype appearance are quite different, and thus need to be matched with specific experimental design and drug efficacy evaluation requirements.
Q: What are the differences between Tub-KO (C001386) and Tubby mice?
A: Tub-KO mice are models of Tub gene knockout, while Tubby mice are models with a spontaneous mutation in the Tub gene. In both models, deficiency of Tub gene leads to protein dysfunction and subsequently photoreceptor cells abnormalities, that cause retinal defects. Stubdal et al. constructed Tub-KO mice through gene knockout, and found almost the same disease phenotype as Tubby mice. Our current results also show that Tub-KO mice have a similar retinal phenotype to Tubby mice.
Q: What are the differences between Rpe65 R44X (C001360) and Rpe65-KO (C001387) mice?
A: Similar to the differences between two RD1 mouse models, Rpe65-KO and Rpe65 R44X mice are models of gene knockout and spontaneous point mutation, respectively. RPE65 protein plays a crucial role in the visual cycle, serving as a key molecule for the conversion and transmission of light signals in the retina. Rpe65 R44X mice with a spontaneous point mutation in the Rpe65 gene have disrupted visual cycle processes, which leads to further degeneration of neural retina and RPE cells, and ultimately irreversible blindness. Rpe65-KO mice are Pde6b gene knockout models. Rpe65 knockout leads to the absence of protein expression, resulting in RPE cell dysfunction, rod photoreceptor apoptosis, disordered outer segment disk arrangements, and extinguished rod photoreceptor waveforms, which cause severe retinal degeneration.
As a comprehensive contract research organization (CRO) solution provider, Cyagen recognizes ophthalmic diseases as a breakthrough point for gene therapy and has established a platform to accelerate translational research of ophthalmic gene therapy. We have equipped the platform with state-of-the-art ophthalmic instruments for small animals and an experienced professional team. With 16 years of gene editing model construction experience, Cyagen can provide you with an array of standardized preclinical research solutions for ophthalmic gene therapy.
Our ophthalmic gene therapy platform is equipped with a full set of state-of-the-art ophthalmic detecting instruments which support a full range of verification services. Our ocular technologies include the Micron IV small animal retinal microscopic imaging system, full-field electroretinogram (ffERG), image-guided optical coherence tomography (OCT) system, and handheld ophthalmotonometer for mice. We can provide detection services for rodent models of eye-related diseases including diabetic retinopathy, retinoblastoma, macular degeneration, pediatric retinopathy of prematurity (ROP), choroidal neovascularization, and retinitis pigmentosa.
With 16 years of experience in the field of custom animal models, Cyagen has independently developed a series of gene editing models targeting ophthalmic diseases (e.g., retinitis pigmentosa (RP), retinal degeneration, Leber congenital amaurosis 2 (LCA2), macular degeneration, Leber congenital amaurosis 10 (LCA10), and endothelial corneal dystrophy). We can provide you with genetically engineered animal models, fully-humanized mouse models, and surgical models to accelerate your preclinical pharmacodynamics evaluations.