Mutations in the retinitis pigmentosa GTPase regulator (RPGR ) gene are associated with X-linked retinitis pigmentosa (XLRP), which accounts for 10% to 20% of all cases of retinitis pigmentosa (RP) and is one of its most severe forms. Herein, we review the research procedure that has culminated in the development of a potential gene therapy for XLRP caused by RPGR mutations.

The author constructed a mouse model of RPGR mutation. After confirming the representative phenotypes of the model by phenotypic analysis, the repair system was delivered to the diseased model mice by rAAV. Homology-directed repair (HDR) was used to demonstrate the good therapeutic effect of AAV-CRISPR/ Cas9-mediated gene editing therapy. The study discovered a gene therapy method for XLRP caused by mutations in the RPGR gene, bringing hope to patients living with this disorder.

1. Foundational research ideas

❖Constructed RPGR knockout (KO) mouse model, this deletion of bases results in premature translation termination, behaving as a null allele. Phenotype analysis revealed photoreceptor degeneration and abnormal expression of photoreceptor proteins.

❖The use of AAV-CRISPR-Pro-mediated gene editing therapy can effectively prevent and treat symptoms such as photoreceptor cell degeneration.

2. Establishment of RPGR knockout mouse model

The author designed two sgRNAs targeting exon 8 of the RPGR gene (Fig. 1A). Then the in vitro transcribed Cas9 mRNA and sgRNA were injected into the fertilized eggs of C57BL/6J mice to construct the RPGR knockout mouse model. And through Sanger sequencing analysis, it was confirmed that the model was successfully constructed (Fig. 1B).

The authors chose mice with a 5 bp deletion (c.974_978delAAATT; p.K325Nfs*1) in exon 8 (Fig. 1C) for next study. The expression and localization of RPGR was analyzed on WT mice, which showed rod-shaped immunoreactivity with brighter puncta at connecting cilia. Whereas, no RPGR staining was detected in RPGR knockout retinas (Fig. 1D). Thus the 5-bp deletion results in inactivation of RPGR.

                                              Figure 1.
The construction and identification of RPGR knockout mouse model

3. Lesion phenotype of RPGR knockout mouse model

The author went on to perform a careful phenotypic analysis of the 5bp deletion mouse model. By performing retinal fundus imaging of WT and RPGR knockout (KO) mice (Fig. 2A), it was found that RPGR KO mice developed multiple small, evenly distributed small yellow-white spots in all quadrants of the retinal fundus at 3 months of age. And as the photoreceptors degenerate, these spots become larger and converge. By 12 months of age, these spots become sparse, but hyperpigmentation is noticeable.

Histologically assessed retinal sections from WT and RPGR KO mice showed a mild senescence-related loss of photoreceptor cells in the outer nuclear layer (ONL) at 6 months of age. By 12 months of age, photoreceptor cell loss in RPGR KO mice became evident (Fig. 2B).

The authors measured ONL thickness and found a significant difference in ONL thickness between WT and mutant mice (Fig. 2C). Along with morphological degeneration, retinal function as measured by Electroretinogram (ERG) also appears abnormal.

Dark-adapted ERG in RPGR KO mice was not significantly different from WT mice at 3 months by ERG, but b-wave amplitude was reduced (Fig. 2D). At 6 months of age, ERG amplitudes (both a- and b-wave amplitudes) decreased further, with virtually no recordable ERG a- and b-wave responses at 12 months of age (Fig. 2E).

                                                          Figure 2. Morphological changes of retina in
RPGR knockout mice

In 6-month-old RPGR knockout (KO) mice, significant reductions in rhodopsin, M-opsin, and S-opsin expression were detected, with only sparse PNA staining visible, which correlates with outer segment integrity and protein stability decline in unison. In 12-month-old RPGR KO mice, PNA and S-opsin staining was further reduced, with only occasional rhodopsin and M-opsin expression observed (Figure 3).

Retinal morphology and function in RPGR KO mice display slow but progressive age-related retinal degeneration. Significant degenerative changes began at 6 months of age, suggesting that the model can mimic the progressive retinal degeneration that occurs in humans. Therefore, this RPGR knockout mouse model provides an excellent animal model system for gene editing therapy research.

                                                    Figure 3.
The degeneration of photoreceptor cells in RPGR knockout mice

4. The retinal degeneration caused by RPGR mutation can be effectively treated by AAV-CRISPR-Pro

The sgRNA expression cassette targeting the mutant RPGR locus and the repair donor template were delivered to 6-month-old RPGR-/yCas9+/WT mice via two separate AAV2/8 vectors (Fig. 4A-D).

                                                                                Figure 4
. Therapy strategy

After 6 months treatment, the number of layers of retinal photoreceptors observed in the treated area was significantly increased compared to the untreated area of the retina of the same eye (layers of photoreceptors in the untreated area: 4 layers  loosely arranged; layers of photoreceptors in the treated area: 9 layers) (Figure 5A, 5B). The ONL in the treated area was significantly thicker than in the untreated area. The density of photoreceptor cells in the treated area was 316 nuclei/100 µm, which was 1.5-fold higher than in the untreated area (128 nuclei/100 µm; Figure 5C). By immunofluorescence analysis, the expression of RPGR, PNA and M-opsin was found to be restored (Fig. 5D).

                                           Figure 5. The retinal structure of the diseased mice recovered after 6 months of treatment

Retinal morphology was assessed at 12 months post-treatment, with RPGR and PNA staining spanning approximately three-quarters of the cross-section (Fig. 6A, 6B). The density of photoreceptor cells in the treated area was 261 nuclei/100 µm, three times that in the untreated area (87 nuclei/100 µm; Figure 6C). These data suggest that CRISPR-Pro-mediated RPGR gene-editing therapy prevents photoreceptor degeneration and that this therapy persists for a long time.

                                  Figure 6. The retinal structure of diseased mice after 12 months get more effective treatment

5. Conclusions

The research in this paper has a clear idea. First, a representative mouse model of progressive retinal degeneration is constructed, phenotypically validated, and then AAV is used to deliver the CRISPR-Pro repair system to the eye for effective treatment. It can be seen that the discovery of therapeutic targets for ophthalmic diseases requires effective disease models. Nowadays, mice are usually used as the object to construct disease models, which is determined by factors such as high homology with human genome, low cost, and mature model construction system. The development of treatment methods generally uses AAV to deliver gene fragments for treatment. Although the strategy of complementing effective gene fragments is commonly used today, CRISPR-Pro repair strategies for gene SNP mutation and other situations are also constantly being researched and developed.

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Hu S, Du J, Chen N, et al. In Vivo CRISPR/Cas9-Mediated Genome Editing Mitigates Photoreceptor Degeneration in a Mouse Model of X-Linked Retinitis Pigmentosa. Invest Ophthalmol Vis Sci. 2020;61(4):31.