CRISPR/Cas9-mediated effective therapy for X-linked retinitis pigmentosa

Mutations in the GTPase regulator (RPGR) gene in retinitis pigmentosa 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 one

1.

❖Constructed RPGR-KO mouse model and found that photoreceptor cells degenerated and abnormal expression of photoreceptor proteins

❖The use of CRISPR/Cas9-mediated gene editing therapy can effectively treat symptoms such as photoreceptor cell degeneration

2.

The authors designed two sgRNAs targeting exon 8 of the RPGR gene (Fig.

The authors chose mice with a 5 bp deletion (c.

Figure 1 Construction and identification of RPGR-KO mouse model

3.

The authors went on to perform a careful phenotypic analysis of the 5bp deletion mouse model

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

The authors measured ONL thickness and found a significant difference in ONL thickness between WT and mutant mice (Fig.

Dark-adapted ERG in RPGR-KO mice was not significantly different from WT mice at 3 months, but b-wave amplitude was reduced by ERG (Fig.

Figure 2 Morphological changes of retina in RPGR-KO mice

In 6-month-old RPGR-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

Retinal morphology and function in RPGR-KO mice display slow but progressive age-related retinal degeneration

Figure 3 Degeneration of photoreceptor cells in RPGR-KO mice

4.

The sgRNA expression cassette targeting the mutant RPGR locus and the repair donor template were delivered to 6-month-old RPGR ^−/y Cas9 ^+/WT mice by two separate AAV2/8 vectors (Fig.

Figure 4 Treatment strategy

Six months after treatment, the number of layers of retinal photoreceptor cells observed in the treated area was significantly increased compared to the untreated area of ​​the retina of the same eye (the number of photoreceptor cell layers in the untreated area: 4 layers, which were loosely arranged; the photoreceptor cell layer in the treated area Number: 9 layers) (Fig.
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

Figure 6 Retinal structure of diseased mice after 12 months

get more effective treatment

V.
Conclusion

The research in this paper has a clear idea.
First, a representative mouse model of progressive retinal degeneration is constructed, and then AAV is used to deliver the CRISPR/Cas9 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 low cost and mature construction system
.
The development of treatment methods generally uses AAV to deliver gene fragments for treatment
.
Although strategies for replenishing effective gene segments are commonly used today, CRISPR/Cas9 repair strategies for gene SNP mutation and other situations are also being continuously 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.