B6-hRHO-P23H Mice

Figure 1. Diagram of the gene editing strategy for the generation of B6-hRHO-P23H mice. The sequences from the ATG start codon to ~500bp downstream of the endogenous mouse Rho gene was replaced with the sequences from the ATG start codon to ~500bp downstream of the human RHO gene and the point mutation p.P23H (CCC to CAC) was introduced into exon1 of human RHO gene.

● Research on retinitis pigmentosa (RP);

● Research on congenital stationary night blindness (CSNB);

● Research on other retinal diseases.

1. Abnormal retinal phenotypes in B6J-hRHO-P23H mice

Figure 2. Fundus morphology, OCT, FFA, and H&E staining results of 8-week-old wild-type mice, B6J-hRHO mice, and 4-week-old heterozygous B6J-hRHO-P23H mice. The results showed that the fundus morphology and retinal morphology of wild-type and B6J-hRHO mice were normal. The fundus vessels of heterozygous B6J-hRHO-P23H mice (HET, i.e., hRHOWT/P23H) were also normal. OCT and H&E staining results showed that the outer nuclear layer (ONL) of the retina was thinner and the overall thickness of the retina was thinner in heterozygous B6J-hRHO-P23H mice than in wild-type and B6J-hRHO mice.

 

2. Abnormalities in electroretinogram (ERG) in B6J-hRHO-P23H mice

Figure 3. Electroretinogram (ERG) of 4-week-old wild-type, B6J-hRHO, and heterozygous B6J-hRHO-P23H mice. The results showed that compared with wild-type and B6J-hRHO mice, the amplitudes of the scotopic a-wave and scotopic b-wave of 4-week-old heterozygous B6J-hRHO-P23H mice were significantly reduced. In contrast, the amplitudes of the photopic a-wave and photopic b-wave did not show a significant decrease.

 

3. Nucleic acid drugs targeting the RHO gene can improve retinal lesions in B6-hRHO-P23H mice

a. Fundus morphology and optical coherence tomography (OCT)

Figure 4. Fundus morphology and OCT results before and after ASO drug treatment in heterozygous B6-hRHO-P23H mice. Based on the publicly available sequence and modification information of the ASO drug QR-1123*, which is used to treat retinitis pigmentosa (RP), a similar antisense oligonucleotide (ASO) was synthesized by GenScript with a structure and function akin to QR-1123. ASO treatment was administered to the mice via bilateral intravitreal injection (dose: 50 μg/μL, 2 μL/eye). The changes in retinal thickness were observed by fundus morphology and OCT on days 0, 7, and 14. The results revealed that compared to the control group (PBS-treated), the ASO-treated mice exhibited significantly increased retinal thickness and a lower ratio of thickness reduction.
*QR-1123, developed by ProQR, is an antisense oligonucleotide drug used to treat autosomal dominant retinitis pigmentosa (adRP) caused by the RHO^P23H mutation. This drug specifically silences mutant mRNA through an RNase H-mediated cleavage mechanism without affecting normal RHO mRNA ^[13].

 

b. Electroretinogram (ERG) test

Figure 5. ERG results before and after ASO drug treatment in heterozygous B6-hRHO-P23H mice. The results indicate that, compared to the control group (PBS-treated), the ASO-treated mice showed significant increases in both scotopic a-wave and b-wave amplitudes on days 7 and 14 after ASO treatment. Notably, on day 14, the scotopic a-wave in the ASO-treated mice was significantly higher than in the PBS-treated group.

1. Basic information about the RHO gene

https://rddc.tsinghua-gd.org/en/gene/6010

 

2. RHO Clinical Variants

The human RHO gene, located on chromosome 5, encodes rhodopsin, the first mutant protein identified in retinitis pigmentosa, a G protein-coupled receptor located in the outer segment of the optic rod cell that is essential for light signaling. Most RHO mutations result in high levels of rhodopsin expression in photoreceptor cells, allowing a large number of mutant proteins to localize abnormally and accumulate in the cell, causing apoptosis of photoreceptor cells that are unable to perform normal light signaling functions.

More than 150 RHO mutations have been identified to be associated with dominant RP, with missense mutations predominating.RHO mutations have the highest mutation rate in European and American populations, especially in the U.S., and a lower rate in Asian populations.P23H was the first RHO mutation to be identified, accounting for approximately 12% of patients in the U.S., while it is rarely found in patients from other national populations^[4]. The P23H mutation in the RHO gene causes the protein to fail to fold correctly, resulting in endoplasmic reticulum stress and cytotoxicity, which ultimately leads to optic rod cell degeneration. The 347th amino acid site at the end of Rhodopsin is another hot spot for mutations, with six different mutation types: P347T, P347A, P347S, P347Q, P347L, and P347R. Among them, P347L is the most common, with a mutation rate of 3.6% in dominant RP, second only to P23H, and studies have reported P347L as a more common mutation site in China^[5]. Regarding the function of the non-coding region, pathogenic mutations (g.3811A>G and g.5167G>T) in the introns of RHO have been reported in the literature and lead to abnormal pre-mRNA shearing^[6].

Current gene therapies targeting the RHO gene for retinitis pigmentosa include ASO and CRISPR, and the use of fully humanized animal models could help drive the further translation of potential RHO-related therapies to clinical trials. clinical trials of ProQR's ASO drug QR-1123 for the treatment of dominant RP associated with the RHO (P23H) mutation are underway (ClinicalTrials.gov ID: NCT04123626), QR-1123 treats disease by inhibiting the production of mutant proteins, and ProQR is using transgenic rats with randomized insertions of RHO (P23H) as a disease model in its development^[7]. Editas announced its in vivo gene editing therapy EDIT-103 for the treatment of RP due to RHO mutations^[8], EDIT-103 is a mutation-independent CRISPR/Cas9-based gene editing therapy that can be administered by subretinal injection by delivering an AAV5 vector containing both knockout and replacement mutants in the Rhodopsin gene to maintain photoreceptor function, which is expected to address more than 150 RHO mutations causing RP. animal models used in studies related to CRISPR therapies in the literature include P23H transgenic mice, P347S transgenic mice, hRHO (C110R/WT) humanized mice, and humanized mouse models with multiple mutations at the same time^[9-12]. In CRISPR therapies, disease models carrying human disease-causing mutated genes are important common factors and humanized disease models are the best validation models before CRISPR therapies go to the clinic.

In summary, the RHO gene is an important pathogenic gene in retinitis pigmentosa with complex pathogenesis and great clinical and genetic heterogeneity. The mutations are predominantly missense mutations, and some of the pathogenic mutations are located in introns, which can lead to abnormal shearing. The B6-hRHO mice and the B6-hRHO-P23H can be applied to preclinical studies of RP gene therapy, and customized models are also available in Cyagen for different point mutations.