Figure 1. Diagram of the gene editing strategy for the generation of B6J-hRHO 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.

● Research on retinitis pigmentosa (RP);

● Research on congenital stationary night blindness (CSNB);

● Research on other retinal diseases.

1.Expression of human RHO gene in B6J-hRHO mice

Figure 2. Detection of human RHO gene expression in B6J-hRHO mice and wild-type mice. The expression of the human RHO gene in mice was detected using qPCR primers for the human RHO gene, and the results showed that B6J-hRHO mice successfully expressed the human RHO gene in vivo compared with wild-type control (B6J).

2.Retinal morphology and retinal vasculature in B6J-hRHO mice

Figure 3. Fundus and retina morphology of 10-week-old homozygous B6J-hRHO mice and C57BL/6J wild-type mice. The fundus morphology (Fundus), retinal optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) results of homozygous B6J-hRHO mice were consistent with those of wild-type mice, indicating that the B6J-hRHO mice had no significant changes in ocular and retinal morphology and were able to maintain normal retinal vascularity and blood circulation.
(WT: wild-type, HO: homozygote).

3.Retinal structure and Rhodopsin expression in B6J-hRHO mice

Figure 4. Detection of retinal structure and Rhodopsin expression in 10-week-old homozygous B6J-hRHO mice and C57BL/6J wild-type mice. The retinal structure and Rhodopsin protein of B6J-hRHO mice and C57BL/6J wild-type mice were detected by H&E staining and immunofluorescence (IF) staining respectively, and the results showed that B6J-hRHO mice had normal retinal structure and Rhodopsin protein expression.

4.Electroretinogram (ERG) testing in B6J-hRHO mice

Figure 5. Electroretinogram (ERG) detection results of homozygous B6J-hRHO mice and C57BL/6J wild-type mice. The amplitudes of a-wave and b-wave in Scotopic and Photopic ERG of homozygous B6J-hRHO mice (RHO) were almost consistent with those of the wild-type (C57BL/6J), indicating that the retinal photoreceptor function of this model mouse was normal.

B6J-RHO-P23H Mice

5.The strategy of B6J-hRHO-P23H mice

Figure 6. Diagram of the gene editing strategy for the generation of B6J-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.

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

Figure 7. The Fundus, OCT, and FFA in the 5-month-old C57BL/6J wild-type mice, B6J-hRHO mice, and B6J-hRHO-P23H mice. The C57BL/6J wild-type mice and B6J-hRHO mice showed normal fundus and retinal morphology, and B6J-hRHO-P23H mice carrying human RHO pathogenic mutation P23H constructed based on B6J-hRHO mice showed obvious abnormal fundus morphology, retina, and blood vessels.

1.Basic information about the RHO gene

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^[2]. 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^[3]. 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^[4].

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^[5]. Editas announced it's in vivo gene editing therapy EDIT-103 for the treatment of RP due to RHO mutations^[6], EDIT-103 is a mutation-independent CRISPR/Cas-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^[7-10]. 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 B6J-hRHO mice and the point mutation models constructed based on this humanized model can be applied to preclinical studies of RP gene therapy, and model customization services are also available in Cyagen for different point mutations.