Department of Ophthalmology, Edward S. Harkness Eye Institute, Columbia University Irving Medical Center, New York, NY 10032, USA.
Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80756, Taiwan.
Int J Mol Sci. 2021 Jul 28;22(15):8069. doi: 10.3390/ijms22158069.
Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a model with a knock-in stop cassette in intron 5 using -CRISPR (fficient dditions with sDNA nserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form "functional" CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future -associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.
全色盲的特征是弱视、畏光、眼球震颤和色盲。以前的全色盲动物模型通过基因增强来恢复锥体细胞功能显示出了有前景的结果。然而，诱发恢复的最佳治疗窗口仍然未知。在这里，我们尝试了两轮基因增强，以生成可恢复的全色盲小鼠模型，包括使用 -CRISPR（带有 sDNA 插入的有效添加-CRISPR）和靶向胚胎干细胞（ES 细胞）在 5 号内含子中敲入终止密码子的模型。该模型表明，与正常的 CNGA3 水平相比，仅来自靶向 ES 细胞的纯合子中保留了 20%的 CNGA3 水平。尽管剩余蛋白的百分比很低，但敲入小鼠模型仍继续产生正常的锥体细胞光转导。我们的结果表明，少量正常的 CNGA3 蛋白足以形成“功能性”CNG 通道，并满足适当的锥体细胞光转导的生理需求。因此，可以得出结论，突变 基因座以破坏功能性四聚体 CNG 通道可能最终需要更有效的 STOP 盒来生成可逆转的全色盲小鼠模型。我们的数据也对未来与 相关的全色盲临床试验具有启示意义，其中仅恢复 20%的功能性 CNGA3 蛋白可能足以形成功能性 CNG 通道，从而挽救锥体细胞反应。
