June 6th, 2023 is the 28th National Eye Care Day of China. Do you know the theme of this year's Eye Care Day?

To focus on rare disease research, Cyagen has launched a new column called "Ten Deadly Sins of Rare Diseases", which aims to demonstrate the pathogenesis, preclinical disease models, and gene therapy methods of multiple rare diseases. By exploring the original sins of these rare diseases, we hope to develop more ideal disease models to promote the development of effective treatments for rare diseases. In this special edition focusing on National Eye Care Day, let's talk about a rare eye disease - retinitis pigmentosa (RP) that is caused by RHO gene mutations.

Pathogenic Mechanisms of RHO

Research on RHO began in the 1950s. In 1957, an American biochemist named George Wald discovered that RHO is the photosensitive pigment required for visual response.[1] This finding provided an important foundation for the possibility of visual restoration techniques. RHO mutations are the main reasons that cause retinitis pigmentosa, and most RHO mutations lead to high-level expression of rhodopsin protein in photoreceptor cells, that results in a large amount of mutated protein mis-localizing and aggregating within cells, thus causing photoreceptor cell death by apoptosis and the inability to perform normal light signal transduction functions. Among the RHO mutations, P23H is the most common pathogenic mutation of the gene.

As early as 2000, researchers have transferred the wild-type RHO gene into P23H transgenic rats, which develop retinitis pigmentosa (RP). The study found that gene delivery therapy could significantly slow down the rate of photoreceptor degeneration in P23H transgenic rats.[2] However, can retinitis pigmentosa (RP) caused by RHO gene mutations actually be cured by simply supplementing RHO expression? The answer is obviously no, since for dominant diseases like RHO-adRP, it is also necessary to simultaneously eliminate gene products that are harmful to cells.

Progress in RHO Gene Therapy Research

Let's take a look at how to treat RP through the "1+1=1" approach in RHO-related gene therapy, which means combining two methods for treatment of the same disease.

AAV Delivery

The drug RhoNova, developed by Roche and Spark, treats RP caused by RHO mutations by combining AAV2/5 delivery to suppress endogenous RHO mutant gene expression through shRNA, and also transferring wild-type (WT) RHO to compensate the expression (shRNA+WT-RHO=RP treatment). Preclinical studies have used P23H transgenic mice as models for drug efficacy evaluation.[3]

Earlier preclinical experiments have shown that the inhibitory effect of shRNA was effective in canine disease models and human cells. However, given that the Rho sequence is not conserved in mice[4], it is not ideal to use traditional mouse models for evaluating the efficiency of shRNAs, as well as other RP-related gene therapies, in a way that will be consistent with the efficacy in patients. Since the mouse Rho gene is not consistent with the human RHO gene, results might not be consistent with humans and transgenic mice are not ideal preclinical disease models for studying RP-related gene therapy.

Targeting RHO with CRISPR Therapy

Editas, founded by Dr. Feng Zhang, announced their progress in using in vivo genome editing therapy EDIT-103 to treat RP caused by RHO mutations. EDIT-103 is a mutation-independent CRISPR-Pro-based genome editing therapy that delivers dual AAV5 vectors to knock out the RHO mutation and introduce WT RHO gene to maintain photoreceptor function. This therapy has the potential of addressing over 150 types of RP caused by various RHO mutations. Similarly, it also involves targeted suppression of mutant RHO with Cas9/gRNA  and compensating with WT-RHO (Cas9+WT-RHO=RP).

Notably, in order to screen highly efficient and specific gRNAs in preclinical research, scientists have cleverly chosen to use the humanized RHO mouse model (mRhohRHO/+), in which the mouse Rho gene was replaced in situ by the human RHO gene. Preclinical studies have shown that hRHO is expressed normally in mice, and EDIT-103 exhibits significant knockout and delivery effects in this humanized RHO mouse model[5]. In the case of RP-related gene therapy research, it quickly becomes evident that it is essential for disease models to carry human genes in order to screen for efficient and specific drugs in preclinical research. In other words, humanized mouse models are more ideal for RP-related drug development, similarly to several other rare diseases that don’t share conserved genetic pathology across species.

Next-Generation Humanized Mouse Model Construction Program: HUGO-GTTM

In addition to RP caused by RHO mutation, the pipeline disclosed by Editas also includes mutations of CEP290 (leading to Leber congenital amaurosis type 10), USH2A (leading to Usher syndrome), SERPINA1 (leading to alpha-1 antitrypsin deficiency), and CFTR (leading to cystic fibrosis). All of these disease pipelines utilize humanized mice as disease models for preclinical studies.

To accelerate new drug development, the "Next-Generation Humanized Mouse Model Construction Program" HUGO-GT™ Program was launched (HUGO-GT: Humanized Genomic Ortholog for Gene Therapy). Cyagen has utilized the TurboKnockout and BAC fusion technologies to replace large sections of the genome, creating a more suitable genomic DNA humanized model for disease research and gene therapy drug development. Based on the Rare Disease Data Center (RDDC), a comprehensive data platform that provides one-stop access to disease-gene-animal model-drug clinical information, we have begun to develop optimized humanized models for rare diseases as part of the HUGO-GT™ Program.

In the field of ophthalmic diseases, Cyagen has developed several humanized mouse models
. For example, we have successfully constructed RHO mice with whole humanized genomic ortholog DNA , as well as their counterparts which carry the P23H point mutation. Other ophthalmic models were also developed, such as hCEP290, hVEGFA, hRPE65, and more. These genomically humanized models are valuable preclinical tools for understanding disease pathology and screening potential therapeutic candidates more consistently with anticipated patient efficacy

Disease Target Gene Target type
Pigmentary degeneration of retina Rho KO, CKO, Humanization (WT, MU)
Mertk KO, CKO
Rpgr KO, CKO
Crb1 KO, CKO
Rd1(Pde6b) MU, KO
Rd10(Pde6b) MU
Macular degeneration Vegfa Humanization(KI, TG)
Abca4(Abcr) KO, CKO, Humanization
Late-Onset Retinal Degeneration Prph2 KO, CKO
Leber congenital amaurosis type 2 Rpe65 KO, MU
Leber congenital amaurosis type 4 Aipl1 KO, CKO
Leber congenital amaurosis type 10 Cep290 KO, CKO, Humanization (WT、MU)
Leber congenital amaurosis type 13 Rdh12 KO, CKO
Achromatopsia Cnga3 KO, CKO
Fuchs endothelial dystrophy Tcf4 KO, CKO, Humanization
Congenital aniridia Pax6 KO, CKO
Choroideremia  Chm KO, CKO
Usher Syndrome Ush2A Humanization (WT, MU)
Vitelliform macular degeneration Best1 KO, CKO
X-linked retinoschisis Rs1 KO, CKO
Oculocutaneous albinism type 1 Tyr CKO
Oculocutaneous albinism type 3 Tyrp1 KO, CKO
Ocular albinism Gpr143 KO, CKO
Bietti crystalline dystrophy Cyp4v3 KO, CKO
Corneal dystrophy Tgfbi KO, CKO, MU, Humanization
Wolfram Syndrome Wfs1 KO, CKO
Pseudoxanthoma elasticum Abcc6 KO, CKO
Retinal pigmentary degeneration, Congenital albinism. Tub KO, CKO
Enhanced S-cone syndrome Nr2e3 KO, CKO



  • Wald G . THE MOLECULAR BASIS OF VISUAL EXCITATION[J]. American Scientist, 1954, 42(1):73-95.
  • Lavail M M ,  Yasumura D ,  Matthes M T , et al. Ribozyme rescue of photoreceptor cells in P23H transgenic rats: Long-term survival and late-stage therapy[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(21):11488-11493.
  • Meng D ,  Ragi S D ,  Tsang S H . Therapy in Rhodopsin-Mediated Autosomal Dominant Retinitis Pigmentosa[J]. Molecular Therapy, 2020, 28(10).
  • Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector[J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(36):E8547-E8556.
  • https://ir.editasmedicine.com/news-releases/news-release-details/editas-medicine-presents-preclinical-data-edit-103-rhodopsin-0