Maddalena A, Kleinlogel S
Institute of Physiology, University of Bern, Bern, Switzerland.
Department of Biomedical Research (DBMR), University of Bern, Bern, Switzerland.
Front Drug Deliv. 2023 Mar 27;3:934394. doi: 10.3389/fddev.2023.934394. eCollection 2023.
Retinitis pigmentosa, an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration of cones, leading to blindness. However, the remaining neural portion of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time. A possible treatment to restore the light sensitivity of the retina consists of rendering the remaining retinal cells photosensitive using optogenetic tools like, for example, Opto-mGluR6, a light-sensitive mGluR6 receptor. We have previously demonstrated that AAV vector-mediated expression of Opto-mGluR6 in ON-bipolar cells restores visual function in otherwise blind mice. However, classical gene supplementation therapy still suffers from high off-target expression rates and uncontrollable target gene expression levels that may lead to either cytotoxicity or lack of functional restoration. To address these issues and achieve cell-specific and endogenously controlled Opto-mGluR6 expression, we employed the CRISPR/Cas technology-in particular, homology-independent targeted integration (HITI) and microhomology-dependent targeted integration (MITI)-to knock-in the Opto-mGluR6 gene behind the ON-bipolar cell-specific GRM6 promoter. We compared four Cas systems and show that SpCas9 for HITI and LbCpf1 for MITI are well suited to promoting knock-in. As AAV2-mediated ON-bipolar cell transduction resulted in inefficiency, we evaluated Exo-AAVs as delivery vehicles and found Exo-AAV1 efficient for targeting ON-bipolar cells. We demonstrate that intravitreal injection of Exo-AAV1 carrying vectors that promote MITI significantly improved visual acuity in otherwise blind mice. We conclude by confirming and providing a qualitative evaluation of the MITI-mediated knock-in in the correct genomic locus.
视网膜色素变性是一种遗传性视网膜变性疾病，其特征是视杆细胞逐渐丧失，随后视锥细胞变性，最终导致失明。然而，视网膜剩余的神经部分（双极细胞和神经节细胞）在解剖学和功能上在较长时间内保持完整。一种恢复视网膜光敏感性的可能治疗方法是使用光遗传学工具，如光敏感的代谢型谷氨酸受体6（Opto-mGluR6），使剩余的视网膜细胞变得对光敏感。我们之前已经证明，腺相关病毒（AAV）载体介导的Opto-mGluR6在ON双极细胞中的表达可恢复原本失明小鼠的视觉功能。然而，传统的基因补充疗法仍然存在高脱靶表达率和无法控制的靶基因表达水平的问题，这可能导致细胞毒性或功能恢复不足。为了解决这些问题并实现细胞特异性和内源性控制的Opto-mGluR6表达，我们采用了CRISPR/Cas技术，特别是同源性无关的靶向整合（HITI）和微同源性依赖的靶向整合（MITI），将Opto-mGluR6基因敲入ON双极细胞特异性GRM6启动子之后。我们比较了四种Cas系统，结果表明用于HITI的SpCas9和用于MITI的LbCpf1非常适合促进基因敲入。由于AAV2介导的ON双极细胞转导效率低下，我们评估了外切AAV作为递送载体，并发现外切AAV1对靶向ON双极细胞有效。我们证明，玻璃体内注射携带促进MITI的载体的外切AAV1可显著提高原本失明小鼠的视力。我们通过确认并对MITI介导的在正确基因组位点的基因敲入进行定性评估来得出结论。
